US20110059096A1 - Targeting of Notch3 Receptor Function for Cancer Therapy - Google Patents
Targeting of Notch3 Receptor Function for Cancer Therapy Download PDFInfo
- Publication number
- US20110059096A1 US20110059096A1 US12/896,439 US89643910A US2011059096A1 US 20110059096 A1 US20110059096 A1 US 20110059096A1 US 89643910 A US89643910 A US 89643910A US 2011059096 A1 US2011059096 A1 US 2011059096A1
- Authority
- US
- United States
- Prior art keywords
- cell
- notch3
- seq
- cells
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000001760 Notch3 Receptor Human genes 0.000 title claims abstract description 195
- 108010029756 Notch3 Receptor Proteins 0.000 title claims abstract description 194
- 230000008685 targeting Effects 0.000 title description 10
- 238000011275 oncology therapy Methods 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 83
- 206010028980 Neoplasm Diseases 0.000 claims description 121
- 201000011510 cancer Diseases 0.000 claims description 45
- 230000011664 signaling Effects 0.000 claims description 24
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 137
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 87
- 239000002246 antineoplastic agent Substances 0.000 abstract description 3
- 238000002648 combination therapy Methods 0.000 abstract description 3
- 230000001093 anti-cancer Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 392
- 108090000623 proteins and genes Proteins 0.000 description 236
- 239000013598 vector Substances 0.000 description 109
- 230000014509 gene expression Effects 0.000 description 102
- 150000007523 nucleic acids Chemical class 0.000 description 99
- 102000004169 proteins and genes Human genes 0.000 description 86
- 235000018102 proteins Nutrition 0.000 description 76
- 108010070047 Notch Receptors Proteins 0.000 description 74
- 102000005650 Notch Receptors Human genes 0.000 description 72
- 102000039446 nucleic acids Human genes 0.000 description 62
- 108020004707 nucleic acids Proteins 0.000 description 62
- 239000000203 mixture Substances 0.000 description 56
- 230000037361 pathway Effects 0.000 description 50
- 241000701161 unidentified adenovirus Species 0.000 description 50
- 241000700605 Viruses Species 0.000 description 43
- 239000003795 chemical substances by application Substances 0.000 description 42
- 108020004414 DNA Proteins 0.000 description 39
- 241000282414 Homo sapiens Species 0.000 description 33
- 239000003446 ligand Substances 0.000 description 33
- 229920001184 polypeptide Polymers 0.000 description 33
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 30
- 241001465754 Metazoa Species 0.000 description 30
- 230000004913 activation Effects 0.000 description 30
- 230000005764 inhibitory process Effects 0.000 description 29
- 208000020816 lung neoplasm Diseases 0.000 description 28
- 108091028043 Nucleic acid sequence Proteins 0.000 description 27
- 235000001014 amino acid Nutrition 0.000 description 27
- 210000001519 tissue Anatomy 0.000 description 27
- 230000006907 apoptotic process Effects 0.000 description 26
- 102000005962 receptors Human genes 0.000 description 26
- 108020003175 receptors Proteins 0.000 description 26
- 229940024606 amino acid Drugs 0.000 description 25
- 150000001413 amino acids Chemical class 0.000 description 25
- 239000003623 enhancer Substances 0.000 description 25
- 238000004519 manufacturing process Methods 0.000 description 25
- 238000013518 transcription Methods 0.000 description 25
- 230000035897 transcription Effects 0.000 description 25
- 230000000694 effects Effects 0.000 description 23
- 201000005202 lung cancer Diseases 0.000 description 23
- 238000011282 treatment Methods 0.000 description 23
- 210000004072 lung Anatomy 0.000 description 22
- 230000006870 function Effects 0.000 description 20
- 208000015181 infectious disease Diseases 0.000 description 20
- 238000004806 packaging method and process Methods 0.000 description 20
- 230000001225 therapeutic effect Effects 0.000 description 20
- 239000013603 viral vector Substances 0.000 description 20
- 230000003612 virological effect Effects 0.000 description 20
- 238000002360 preparation method Methods 0.000 description 19
- 239000000047 product Substances 0.000 description 19
- 238000012384 transportation and delivery Methods 0.000 description 19
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 18
- 230000001419 dependent effect Effects 0.000 description 18
- 239000003540 gamma secretase inhibitor Substances 0.000 description 18
- 238000001727 in vivo Methods 0.000 description 18
- 238000000746 purification Methods 0.000 description 18
- 230000010076 replication Effects 0.000 description 18
- 238000012546 transfer Methods 0.000 description 18
- 230000027455 binding Effects 0.000 description 17
- 239000002502 liposome Substances 0.000 description 17
- 230000001105 regulatory effect Effects 0.000 description 17
- 241001430294 unidentified retrovirus Species 0.000 description 17
- 102000043136 MAP kinase family Human genes 0.000 description 16
- 108091054455 MAP kinase family Proteins 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 16
- 239000003550 marker Substances 0.000 description 16
- 230000001177 retroviral effect Effects 0.000 description 16
- 241000894007 species Species 0.000 description 16
- 241000700584 Simplexvirus Species 0.000 description 15
- 108700019146 Transgenes Proteins 0.000 description 15
- 210000002966 serum Anatomy 0.000 description 15
- 210000002845 virion Anatomy 0.000 description 15
- 229940125373 Gamma-Secretase Inhibitor Drugs 0.000 description 14
- 238000012545 processing Methods 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 13
- 238000000338 in vitro Methods 0.000 description 13
- 230000001404 mediated effect Effects 0.000 description 13
- 230000035772 mutation Effects 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 13
- 102400001368 Epidermal growth factor Human genes 0.000 description 12
- 101800003838 Epidermal growth factor Proteins 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 230000008901 benefit Effects 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 12
- 239000000306 component Substances 0.000 description 12
- 238000012217 deletion Methods 0.000 description 12
- 230000037430 deletion Effects 0.000 description 12
- 229940116977 epidermal growth factor Drugs 0.000 description 12
- 239000013604 expression vector Substances 0.000 description 12
- 230000004927 fusion Effects 0.000 description 12
- 238000001476 gene delivery Methods 0.000 description 12
- 230000002458 infectious effect Effects 0.000 description 12
- 239000003112 inhibitor Substances 0.000 description 12
- 230000008488 polyadenylation Effects 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- 210000004881 tumor cell Anatomy 0.000 description 12
- 108090001090 Lectins Proteins 0.000 description 11
- 102000004856 Lectins Human genes 0.000 description 11
- NKHUILHBYOOZDF-NCOIWELASA-N chembl196215 Chemical compound N1S(=O)(=O)N(CC(F)(F)F)C[C@]21[C@@H]1CC[C@H]2CC2=CC=C(\C=C\CN3CCC(CC3)C(F)(F)F)C=C2C1 NKHUILHBYOOZDF-NCOIWELASA-N 0.000 description 11
- 238000011161 development Methods 0.000 description 11
- 230000018109 developmental process Effects 0.000 description 11
- 238000004520 electroporation Methods 0.000 description 11
- 230000001939 inductive effect Effects 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 239000002523 lectin Substances 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 11
- 125000003729 nucleotide group Chemical group 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 10
- 101000994439 Danio rerio Protein jagged-1a Proteins 0.000 description 10
- 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 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 10
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 10
- 102400000552 Notch 1 intracellular domain Human genes 0.000 description 10
- 101800001628 Notch 1 intracellular domain Proteins 0.000 description 10
- 206010035226 Plasma cell myeloma Diseases 0.000 description 10
- 102100032702 Protein jagged-1 Human genes 0.000 description 10
- 239000000427 antigen Substances 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 238000003776 cleavage reaction Methods 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 230000006698 induction Effects 0.000 description 10
- 201000000050 myeloid neoplasm Diseases 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- 230000007017 scission Effects 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 230000014616 translation Effects 0.000 description 10
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 9
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 9
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 9
- 241000124008 Mammalia Species 0.000 description 9
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 9
- 239000004480 active ingredient Substances 0.000 description 9
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 9
- 230000000295 complement effect Effects 0.000 description 9
- 230000007423 decrease Effects 0.000 description 9
- 229940121647 egfr inhibitor Drugs 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 238000001415 gene therapy Methods 0.000 description 9
- 238000009396 hybridization Methods 0.000 description 9
- 230000002163 immunogen Effects 0.000 description 9
- 230000003993 interaction Effects 0.000 description 9
- 229920001223 polyethylene glycol Polymers 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 9
- 238000002741 site-directed mutagenesis Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000002560 therapeutic procedure Methods 0.000 description 9
- 238000013519 translation Methods 0.000 description 9
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 8
- 108020004635 Complementary DNA Proteins 0.000 description 8
- 108060006698 EGF receptor Proteins 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 8
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 8
- -1 NcoR Proteins 0.000 description 8
- 239000002202 Polyethylene glycol Substances 0.000 description 8
- 241000700159 Rattus Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 8
- 238000001042 affinity chromatography Methods 0.000 description 8
- 229960004316 cisplatin Drugs 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 230000003053 immunization Effects 0.000 description 8
- 229960000310 isoleucine Drugs 0.000 description 8
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 8
- 210000002569 neuron Anatomy 0.000 description 8
- 239000013615 primer Substances 0.000 description 8
- 230000002797 proteolythic effect Effects 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 230000009466 transformation Effects 0.000 description 8
- 239000003981 vehicle Substances 0.000 description 8
- 108010043324 Amyloid Precursor Protein Secretases Proteins 0.000 description 7
- 239000004475 Arginine Substances 0.000 description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 7
- 108020004705 Codon Proteins 0.000 description 7
- 102100034349 Integrase Human genes 0.000 description 7
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 7
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 description 7
- 108020004459 Small interfering RNA Proteins 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 238000007792 addition Methods 0.000 description 7
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 7
- 235000009582 asparagine Nutrition 0.000 description 7
- 229960001230 asparagine Drugs 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 238000010804 cDNA synthesis Methods 0.000 description 7
- 231100000504 carcinogenesis Toxicity 0.000 description 7
- 230000001413 cellular effect Effects 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 230000002950 deficient Effects 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 7
- 239000003102 growth factor Substances 0.000 description 7
- 210000004408 hybridoma Anatomy 0.000 description 7
- 201000005296 lung carcinoma Diseases 0.000 description 7
- 210000003463 organelle Anatomy 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 210000000952 spleen Anatomy 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000004474 valine Substances 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 6
- 230000005778 DNA damage Effects 0.000 description 6
- 231100000277 DNA damage Toxicity 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 102100034428 Dual specificity protein phosphatase 1 Human genes 0.000 description 6
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 6
- 108010081348 HRT1 protein Hairy Proteins 0.000 description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 6
- 241000713666 Lentivirus Species 0.000 description 6
- 239000004472 Lysine Substances 0.000 description 6
- 102400000547 Notch 3 intracellular domain Human genes 0.000 description 6
- 101800000993 Notch 3 intracellular domain Proteins 0.000 description 6
- 102000001759 Notch1 Receptor Human genes 0.000 description 6
- 108010029755 Notch1 Receptor Proteins 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 108700020796 Oncogene Proteins 0.000 description 6
- 241000710960 Sindbis virus Species 0.000 description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 6
- 239000004473 Threonine Substances 0.000 description 6
- 241000700618 Vaccinia virus Species 0.000 description 6
- 208000009956 adenocarcinoma Diseases 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- 210000003719 b-lymphocyte Anatomy 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 230000036952 cancer formation Effects 0.000 description 6
- 230000033026 cell fate determination Effects 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 210000000981 epithelium Anatomy 0.000 description 6
- 210000003527 eukaryotic cell Anatomy 0.000 description 6
- 229960002949 fluorouracil Drugs 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 230000008506 pathogenesis Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 208000024827 Alzheimer disease Diseases 0.000 description 5
- 208000005623 Carcinogenesis Diseases 0.000 description 5
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 5
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 5
- 241001135569 Human adenovirus 5 Species 0.000 description 5
- 241000725303 Human immunodeficiency virus Species 0.000 description 5
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 5
- 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 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 5
- 108700005077 Viral Genes Proteins 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 229940009098 aspartate Drugs 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000000234 capsid Anatomy 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 229930195712 glutamate Natural products 0.000 description 5
- 210000003494 hepatocyte Anatomy 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 230000000977 initiatory effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 201000001441 melanoma Diseases 0.000 description 5
- 229930182817 methionine Natural products 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 5
- 230000005855 radiation Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 210000001550 testis Anatomy 0.000 description 5
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 4
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 4
- 108090000672 Annexin A5 Proteins 0.000 description 4
- 102000004121 Annexin A5 Human genes 0.000 description 4
- 201000009030 Carcinoma Diseases 0.000 description 4
- 230000004543 DNA replication Effects 0.000 description 4
- 101710132784 Dual specificity protein phosphatase 1 Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 101000584743 Homo sapiens Recombining binding protein suppressor of hairless Proteins 0.000 description 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-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
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 206010027476 Metastases Diseases 0.000 description 4
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 4
- 108010050254 Presenilins Proteins 0.000 description 4
- 102000015499 Presenilins Human genes 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 102100030000 Recombining binding protein suppressor of hairless Human genes 0.000 description 4
- 241000713311 Simian immunodeficiency virus Species 0.000 description 4
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 4
- 229940009456 adriamycin Drugs 0.000 description 4
- 235000004279 alanine Nutrition 0.000 description 4
- 125000003275 alpha amino acid group Chemical group 0.000 description 4
- 210000002383 alveolar type I cell Anatomy 0.000 description 4
- 230000033115 angiogenesis Effects 0.000 description 4
- 239000012736 aqueous medium Substances 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 239000001506 calcium phosphate Substances 0.000 description 4
- 229910000389 calcium phosphate Inorganic materials 0.000 description 4
- 235000011010 calcium phosphates Nutrition 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 230000011712 cell development Effects 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 230000005757 colony formation Effects 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 239000002612 dispersion medium Substances 0.000 description 4
- 230000003828 downregulation Effects 0.000 description 4
- 230000008482 dysregulation Effects 0.000 description 4
- 229960005420 etoposide Drugs 0.000 description 4
- 210000003128 head Anatomy 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 210000002510 keratinocyte Anatomy 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 238000012423 maintenance Methods 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000009401 metastasis Effects 0.000 description 4
- 229960000485 methotrexate Drugs 0.000 description 4
- 210000003739 neck Anatomy 0.000 description 4
- 210000004940 nucleus Anatomy 0.000 description 4
- 231100000590 oncogenic Toxicity 0.000 description 4
- 230000002246 oncogenic effect Effects 0.000 description 4
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 4
- 239000002644 phorbol ester Substances 0.000 description 4
- 108700004029 pol Genes Proteins 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000006337 proteolytic cleavage Effects 0.000 description 4
- 210000001938 protoplast Anatomy 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
- 238000001959 radiotherapy Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000006152 selective media Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 230000008093 supporting effect Effects 0.000 description 4
- 238000004114 suspension culture Methods 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- 238000011830 transgenic mouse model Methods 0.000 description 4
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 4
- 230000006459 vascular development Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- AGNGYMCLFWQVGX-AGFFZDDWSA-N (e)-1-[(2s)-2-amino-2-carboxyethoxy]-2-diazonioethenolate Chemical compound OC(=O)[C@@H](N)CO\C([O-])=C\[N+]#N AGNGYMCLFWQVGX-AGFFZDDWSA-N 0.000 description 3
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 3
- 108010060434 Co-Repressor Proteins Proteins 0.000 description 3
- 102000008169 Co-Repressor Proteins Human genes 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000206602 Eukaryota Species 0.000 description 3
- 108010007457 Extracellular Signal-Regulated MAP Kinases Proteins 0.000 description 3
- 108091006020 Fc-tagged proteins Proteins 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 3
- 108010050904 Interferons Proteins 0.000 description 3
- 206010064912 Malignant transformation Diseases 0.000 description 3
- 229930193140 Neomycin Natural products 0.000 description 3
- 230000005913 Notch signaling pathway Effects 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 241000276498 Pollachius virens Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 108020004440 Thymidine kinase Proteins 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 206010046865 Vaccinia virus infection Diseases 0.000 description 3
- 108010067390 Viral Proteins Proteins 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 229960003896 aminopterin Drugs 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 238000009175 antibody therapy Methods 0.000 description 3
- 210000000628 antibody-producing cell Anatomy 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- 239000003429 antifungal agent Substances 0.000 description 3
- 230000001640 apoptogenic effect Effects 0.000 description 3
- 238000003491 array Methods 0.000 description 3
- 210000001130 astrocyte Anatomy 0.000 description 3
- 229950011321 azaserine Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 210000000233 bronchiolar non-ciliated Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000008358 core component Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000000551 dentifrice Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 108700004025 env Genes Proteins 0.000 description 3
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 3
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 3
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 108700004026 gag Genes Proteins 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 101150028578 grp78 gene Proteins 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000007918 intramuscular administration Methods 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 239000007951 isotonicity adjuster Substances 0.000 description 3
- 238000011031 large-scale manufacturing process Methods 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 208000037841 lung tumor Diseases 0.000 description 3
- 210000001165 lymph node Anatomy 0.000 description 3
- 230000036212 malign transformation Effects 0.000 description 3
- 230000003211 malignant effect Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000000520 microinjection Methods 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 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 3
- 229960004927 neomycin Drugs 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 208000008443 pancreatic carcinoma Diseases 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 101150088264 pol gene Proteins 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 210000002307 prostate Anatomy 0.000 description 3
- 239000002510 pyrogen Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000008707 rearrangement Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 208000007089 vaccinia Diseases 0.000 description 3
- 230000029812 viral genome replication Effects 0.000 description 3
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 2
- 241000710929 Alphavirus Species 0.000 description 2
- 206010003571 Astrocytoma Diseases 0.000 description 2
- 102100026596 Bcl-2-like protein 1 Human genes 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 102100021984 C-C motif chemokine 4-like Human genes 0.000 description 2
- 102100032912 CD44 antigen Human genes 0.000 description 2
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 2
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 239000012623 DNA damaging agent Substances 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 101150117962 DUSP1 gene Proteins 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- 241000702421 Dependoparvovirus Species 0.000 description 2
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 102100031111 Disintegrin and metalloproteinase domain-containing protein 17 Human genes 0.000 description 2
- 206010013801 Duchenne Muscular Dystrophy Diseases 0.000 description 2
- 108010069091 Dystrophin Proteins 0.000 description 2
- 101150029662 E1 gene Proteins 0.000 description 2
- 108700041152 Endoplasmic Reticulum Chaperone BiP Proteins 0.000 description 2
- 102100021451 Endoplasmic reticulum chaperone BiP Human genes 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- 241000713730 Equine infectious anemia virus Species 0.000 description 2
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 102100021186 Granulysin Human genes 0.000 description 2
- 101150112743 HSPA5 gene Proteins 0.000 description 2
- 208000031220 Hemophilia Diseases 0.000 description 2
- 208000009292 Hemophilia A Diseases 0.000 description 2
- 108091027305 Heteroduplex Proteins 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- 101710168537 High mobility group protein B1 Proteins 0.000 description 2
- 101100118545 Holotrichia diomphalia EGF-like gene Proteins 0.000 description 2
- 101000777471 Homo sapiens C-C motif chemokine 4 Proteins 0.000 description 2
- 101000896959 Homo sapiens C-C motif chemokine 4-like Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101001040751 Homo sapiens Granulysin Proteins 0.000 description 2
- 101000577202 Homo sapiens Neurogenic locus notch homolog protein 3 Proteins 0.000 description 2
- 101001042049 Human herpesvirus 1 (strain 17) Transcriptional regulator ICP22 Proteins 0.000 description 2
- 101000999690 Human herpesvirus 2 (strain HG52) E3 ubiquitin ligase ICP22 Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 102000003996 Interferon-beta Human genes 0.000 description 2
- 108090000467 Interferon-beta Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- ZQISRDCJNBUVMM-UHFFFAOYSA-N L-Histidinol Natural products OCC(N)CC1=CN=CN1 ZQISRDCJNBUVMM-UHFFFAOYSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- ZQISRDCJNBUVMM-YFKPBYRVSA-N L-histidinol Chemical compound OC[C@@H](N)CC1=CNC=N1 ZQISRDCJNBUVMM-YFKPBYRVSA-N 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- 102000043131 MHC class II family Human genes 0.000 description 2
- 108091054438 MHC class II family Proteins 0.000 description 2
- 101150012093 MKP1 gene Proteins 0.000 description 2
- 108010059343 MM Form Creatine Kinase Proteins 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000003792 Metallothionein Human genes 0.000 description 2
- 108090000157 Metallothionein Proteins 0.000 description 2
- 206010054949 Metaplasia Diseases 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 108010008707 Mucin-1 Proteins 0.000 description 2
- 102000007298 Mucin-1 Human genes 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 241000714177 Murine leukemia virus Species 0.000 description 2
- 101150103623 NOTCH3 gene Proteins 0.000 description 2
- 108700019961 Neoplasm Genes Proteins 0.000 description 2
- 102000048850 Neoplasm Genes Human genes 0.000 description 2
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 description 2
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 2
- 241001452677 Ogataea methanolica Species 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 102000037602 Platelet Endothelial Cell Adhesion Molecule-1 Human genes 0.000 description 2
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 2
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 2
- 108010071690 Prealbumin Proteins 0.000 description 2
- 102000007584 Prealbumin Human genes 0.000 description 2
- 208000009052 Precursor T-Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102100038358 Prostate-specific antigen Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 102100032733 Protein jagged-2 Human genes 0.000 description 2
- 101710170213 Protein jagged-2 Proteins 0.000 description 2
- 102000007615 Pulmonary Surfactant-Associated Protein A Human genes 0.000 description 2
- 108010007100 Pulmonary Surfactant-Associated Protein A Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 241001068263 Replication competent viruses Species 0.000 description 2
- 101100111629 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) KAR2 gene Proteins 0.000 description 2
- 101100478343 Schizosaccharomyces pombe (strain 972 / ATCC 24843) srk1 gene Proteins 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 102000054727 Serum Amyloid A Human genes 0.000 description 2
- 108700028909 Serum Amyloid A Proteins 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 208000024313 Testicular Neoplasms Diseases 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000006601 Thymidine Kinase Human genes 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 108010003205 Vasoactive Intestinal Peptide Proteins 0.000 description 2
- 102400000015 Vasoactive intestinal peptide Human genes 0.000 description 2
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 239000003070 absorption delaying agent Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 210000002588 alveolar type II cell Anatomy 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 2
- 230000001028 anti-proliverative effect Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 108010006025 bovine growth hormone Proteins 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004900 c-terminal fragment Anatomy 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 2
- 229940127093 camptothecin Drugs 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000006552 constitutive activation Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 229960000640 dactinomycin Drugs 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 108020001096 dihydrofolate reductase Proteins 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 2
- 230000005014 ectopic expression Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 101150030339 env gene Proteins 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 101150098622 gag gene Proteins 0.000 description 2
- 102000038383 gamma-secretases Human genes 0.000 description 2
- 108091007739 gamma-secretases Proteins 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
- 108010017007 glucose-regulated proteins Proteins 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000006882 induction of apoptosis Effects 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000002601 intratumoral effect Effects 0.000 description 2
- VBUWHHLIZKOSMS-RIWXPGAOSA-N invicorp Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)C(C)C)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 VBUWHHLIZKOSMS-RIWXPGAOSA-N 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 101710130522 mRNA export factor Proteins 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 235000009973 maize Nutrition 0.000 description 2
- 210000002752 melanocyte Anatomy 0.000 description 2
- 230000015689 metaplastic ossification Effects 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004001 molecular interaction Effects 0.000 description 2
- 239000002324 mouth wash Substances 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 210000005170 neoplastic cell Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 210000004412 neuroendocrine cell Anatomy 0.000 description 2
- 238000007500 overflow downdraw method Methods 0.000 description 2
- 239000006072 paste Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 235000020030 perry Nutrition 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 2
- 239000002987 primer (paints) Substances 0.000 description 2
- 230000001566 pro-viral effect Effects 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 210000004116 schwann cell Anatomy 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 description 2
- 230000037351 starvation Effects 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 238000012301 transgenic model Methods 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 2
- 230000005747 tumor angiogenesis Effects 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 2
- 108700026220 vif Genes Proteins 0.000 description 2
- 230000006648 viral gene expression Effects 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- CJDRUOGAGYHKKD-XMTJACRCSA-N (+)-Ajmaline Natural products O[C@H]1[C@@H](CC)[C@@H]2[C@@H]3[C@H](O)[C@@]45[C@@H](N(C)c6c4cccc6)[C@@H](N1[C@H]3C5)C2 CJDRUOGAGYHKKD-XMTJACRCSA-N 0.000 description 1
- QGVLYPPODPLXMB-UBTYZVCOSA-N (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-4a,7b,9,9a-tetrahydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-1,1a,1b,4,4a,7a,7b,8,9,9a-decahydro-5H-cyclopropa[3,4]benzo[1,2-e]azulen-5-one Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)[C@@H](O)[C@@]3(O)C(C)(C)[C@H]3[C@@H]21 QGVLYPPODPLXMB-UBTYZVCOSA-N 0.000 description 1
- VVJYUAYZJAKGRQ-BGZDPUMWSA-N 1-[(2r,4r,5s,6r)-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)C1 VVJYUAYZJAKGRQ-BGZDPUMWSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- SGTNSNPWRIOYBX-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile Chemical compound C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-UHFFFAOYSA-N 0.000 description 1
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- 108091007505 ADAM17 Proteins 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 108010024223 Adenine phosphoribosyltransferase Proteins 0.000 description 1
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 206010001258 Adenoviral infections Diseases 0.000 description 1
- 108700026758 Adenovirus hexon capsid Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102100033312 Alpha-2-macroglobulin Human genes 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 102000004580 Aspartic Acid Proteases Human genes 0.000 description 1
- 108010017640 Aspartic Acid Proteases Proteins 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 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
- 102100037674 Bis(5'-adenosyl)-triphosphatase Human genes 0.000 description 1
- 241000237519 Bivalvia Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- 241000197194 Bulla Species 0.000 description 1
- 101100379376 Caenorhabditis elegans apx-1 gene Proteins 0.000 description 1
- 101100337060 Caenorhabditis elegans glp-1 gene Proteins 0.000 description 1
- 101100074828 Caenorhabditis elegans lin-12 gene Proteins 0.000 description 1
- 102000004414 Calcitonin Gene-Related Peptide Human genes 0.000 description 1
- 108090000932 Calcitonin Gene-Related Peptide Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 244000045232 Canavalia ensiformis Species 0.000 description 1
- 235000010520 Canavalia ensiformis Nutrition 0.000 description 1
- 241000701157 Canine mastadenovirus A Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 241001308924 Cyclorana maini Species 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 239000012624 DNA alkylating agent Substances 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 108700010025 DRD1 Proteins 0.000 description 1
- 101000876610 Dictyostelium discoideum Extracellular signal-regulated kinase 2 Proteins 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 102000010778 Dual Specificity Phosphatase 1 Human genes 0.000 description 1
- 108010038537 Dual Specificity Phosphatase 1 Proteins 0.000 description 1
- 102000001039 Dystrophin Human genes 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 241000710945 Eastern equine encephalitis virus Species 0.000 description 1
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 1
- 102100039328 Endoplasmin Human genes 0.000 description 1
- 102400000792 Endothelial monocyte-activating polypeptide 2 Human genes 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 208000031637 Erythroblastic Acute Leukemia Diseases 0.000 description 1
- 208000036566 Erythroleukaemia Diseases 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 102000015212 Fas Ligand Protein Human genes 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101001053811 Gallus gallus Dorsalin-1 Proteins 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 208000015872 Gaucher disease Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 241000713813 Gibbon ape leukemia virus Species 0.000 description 1
- 102000053171 Glial Fibrillary Acidic Human genes 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 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 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 1
- 108010062347 HLA-DQ Antigens Proteins 0.000 description 1
- 108010067802 HLA-DR alpha-Chains Proteins 0.000 description 1
- 108010024164 HLA-G Antigens Proteins 0.000 description 1
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 1
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 1
- 108010058611 Helix lectin Proteins 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 241000175212 Herpesvirales Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101710155188 Hexon-interlacing protein Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101100005713 Homo sapiens CD4 gene Proteins 0.000 description 1
- 101000632178 Homo sapiens Homeobox protein Nkx-2.1 Proteins 0.000 description 1
- 101100343328 Homo sapiens LIMK2 gene Proteins 0.000 description 1
- 101001020310 Homo sapiens Liprin-alpha-1 Proteins 0.000 description 1
- 101001005664 Homo sapiens Mastermind-like protein 1 Proteins 0.000 description 1
- 101001005667 Homo sapiens Mastermind-like protein 2 Proteins 0.000 description 1
- 101001005668 Homo sapiens Mastermind-like protein 3 Proteins 0.000 description 1
- 101001052493 Homo sapiens Mitogen-activated protein kinase 1 Proteins 0.000 description 1
- 101000978766 Homo sapiens Neurogenic locus notch homolog protein 1 Proteins 0.000 description 1
- 101000612671 Homo sapiens Pulmonary surfactant-associated protein C Proteins 0.000 description 1
- 101000845269 Homo sapiens Transcription termination factor 1 Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241000598171 Human adenovirus sp. Species 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 101100195053 Human herpesvirus 1 (strain 17) RIR1 gene Proteins 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 241000282620 Hylobates sp. Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 1
- 101150090364 ICP0 gene Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 108020005350 Initiator Codon Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 1
- 102400000022 Insulin-like growth factor II Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 102100020873 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102100040445 Keratin, type I cytoskeletal 14 Human genes 0.000 description 1
- 108010066321 Keratin-14 Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 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
- 208000032420 Latent Infection Diseases 0.000 description 1
- 240000004322 Lens culinaris Species 0.000 description 1
- 235000014647 Lens culinaris subsp culinaris Nutrition 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 102100035684 Liprin-alpha-1 Human genes 0.000 description 1
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 1
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 102100025129 Mastermind-like protein 1 Human genes 0.000 description 1
- 102100025130 Mastermind-like protein 2 Human genes 0.000 description 1
- 102100025134 Mastermind-like protein 3 Human genes 0.000 description 1
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 101710201349 Metallothionein B Proteins 0.000 description 1
- 102100031347 Metallothionein-2 Human genes 0.000 description 1
- 101710094505 Metallothionein-2 Proteins 0.000 description 1
- 102000004182 Mitogen-Activated Protein Kinase Phosphatases Human genes 0.000 description 1
- 108010082747 Mitogen-Activated Protein Kinase Phosphatases Proteins 0.000 description 1
- 206010073148 Multiple endocrine neoplasia type 2A Diseases 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 101100007124 Mus musculus Col11a2 gene Proteins 0.000 description 1
- 101100444898 Mus musculus Egr1 gene Proteins 0.000 description 1
- 101100348845 Mus musculus Notch3 gene Proteins 0.000 description 1
- 101100317378 Mus musculus Wnt3 gene Proteins 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 206010029113 Neovascularisation Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 102000001068 Neural Cell Adhesion Molecules Human genes 0.000 description 1
- 102100023616 Neural cell adhesion molecule L1-like protein Human genes 0.000 description 1
- 206010052399 Neuroendocrine tumour Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 102000006570 Non-Histone Chromosomal Proteins Human genes 0.000 description 1
- 108010008964 Non-Histone Chromosomal Proteins Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 102100030569 Nuclear receptor corepressor 2 Human genes 0.000 description 1
- 101710153660 Nuclear receptor corepressor 2 Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 241001504519 Papio ursinus Species 0.000 description 1
- 241000237988 Patellidae Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010029182 Pectin lyase Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 108010076039 Polyproteins Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 208000017414 Precursor T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 108010015078 Pregnancy-Associated alpha 2-Macroglobulins Proteins 0.000 description 1
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 1
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 1
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 101000868151 Rattus norvegicus Somatotropin Proteins 0.000 description 1
- 241000702263 Reovirus sp. Species 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 240000000528 Ricinus communis Species 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 241000710961 Semliki Forest virus Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102000011842 Serrate-Jagged Proteins Human genes 0.000 description 1
- 108010036039 Serrate-Jagged Proteins Proteins 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 101100289792 Squirrel monkey polyomavirus large T gene Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 208000029052 T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 102100031079 Transcription termination factor 1 Human genes 0.000 description 1
- 102000013394 Troponin I Human genes 0.000 description 1
- 108010065729 Troponin I Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 102100039094 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 241000710959 Venezuelan equine encephalitis virus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 102100035071 Vimentin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 108010087302 Viral Structural Proteins Proteins 0.000 description 1
- 241000710951 Western equine encephalitis virus Species 0.000 description 1
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- VRGWBRLULZUWAJ-XFFXIZSCSA-N [(2s)-2-[(1r,3z,5s,8z,12z,15s)-5,17-dihydroxy-4,8,12,15-tetramethyl-16-oxo-18-bicyclo[13.3.0]octadeca-3,8,12,17-tetraenyl]propyl] acetate Chemical compound C1\C=C(C)/CC\C=C(C)/CC[C@H](O)\C(C)=C/C[C@@H]2C([C@@H](COC(C)=O)C)=C(O)C(=O)[C@]21C VRGWBRLULZUWAJ-XFFXIZSCSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 239000003082 abrasive agent Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000021841 acute erythroid leukemia Diseases 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 101150087698 alpha gene Proteins 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000011394 anticancer treatment Methods 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
- 239000003443 antiviral agent Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 239000013602 bacteriophage vector Substances 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 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
- WPIHMWBQRSAMDE-YCZTVTEBSA-N beta-D-galactosyl-(1->4)-beta-D-galactosyl-N-(pentacosanoyl)sphingosine Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC(=O)N[C@@H](CO[C@@H]1O[C@H](CO)[C@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O)[C@H](O)\C=C\CCCCCCCCCCCCC WPIHMWBQRSAMDE-YCZTVTEBSA-N 0.000 description 1
- 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 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 108091007737 beta-secretases Proteins 0.000 description 1
- 239000003012 bilayer membrane Substances 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 108010005713 bis(5'-adenosyl)triphosphatase Proteins 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 210000003123 bronchiole Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 210000003837 chick embryo Anatomy 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- BFPSDSIWYFKGBC-UHFFFAOYSA-N chlorotrianisene Chemical compound C1=CC(OC)=CC=C1C(Cl)=C(C=1C=CC(OC)=CC=1)C1=CC=C(OC)C=C1 BFPSDSIWYFKGBC-UHFFFAOYSA-N 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 235000020639 clam Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000112 colonic effect Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 229940046044 combinations of antineoplastic agent Drugs 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000030944 contact inhibition Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009133 cooperative interaction Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000021040 cytoplasmic transport Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000003110 dot immunobinding assay Methods 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 210000003027 ear inner Anatomy 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000000408 embryogenic effect Effects 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- YJGVMLPVUAXIQN-UHFFFAOYSA-N epipodophyllotoxin Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YJGVMLPVUAXIQN-UHFFFAOYSA-N 0.000 description 1
- 210000005081 epithelial layer Anatomy 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000004088 foaming agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- VRGWBRLULZUWAJ-UHFFFAOYSA-N fusaproliferin Natural products C1C=C(C)CCC=C(C)CCC(O)C(C)=CCC2C(C(COC(C)=O)C)=C(O)C(=O)C21C VRGWBRLULZUWAJ-UHFFFAOYSA-N 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 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
- 238000003197 gene knockdown Methods 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000009395 genetic defect Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 231100000734 genotoxic potential Toxicity 0.000 description 1
- 210000001703 glandular epithelial cell Anatomy 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000003067 hemagglutinative effect Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 102000045609 human NOTCH1 Human genes 0.000 description 1
- 102000047087 human SFTPC Human genes 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 108010034897 lentil lectin Proteins 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- YAFQFNOUYXZVPZ-UHFFFAOYSA-N liproxstatin-1 Chemical compound ClC1=CC=CC(CNC=2C3(CCNCC3)NC3=CC=CC=C3N=2)=C1 YAFQFNOUYXZVPZ-UHFFFAOYSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000007040 lung development Effects 0.000 description 1
- 230000007905 lung morphogenesis Effects 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 208000003747 lymphoid leukemia Diseases 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 108010000525 member 1 small inducible cytokine subfamily E Proteins 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 229940051866 mouthwash Drugs 0.000 description 1
- 206010051747 multiple endocrine neoplasia Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 101150008049 mx gene Proteins 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 230000001114 myogenic effect Effects 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 210000004898 n-terminal fragment Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 230000010309 neoplastic transformation Effects 0.000 description 1
- 230000000955 neuroendocrine Effects 0.000 description 1
- 208000016065 neuroendocrine neoplasm Diseases 0.000 description 1
- 201000011519 neuroendocrine tumor Diseases 0.000 description 1
- 230000004766 neurogenesis Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000004145 nucleotide salvage Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 229940127084 other anti-cancer agent Drugs 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 210000002741 palatine tonsil Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 238000004810 partition chromatography Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 230000009038 pharmacological inhibition Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 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@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[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 CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- QGVLYPPODPLXMB-QXYKVGAMSA-N phorbol Natural products C[C@@H]1[C@@H](O)[C@]2(O)[C@H]([C@H]3C=C(CO)C[C@@]4(O)[C@H](C=C(C)C4=O)[C@@]13O)C2(C)C QGVLYPPODPLXMB-QXYKVGAMSA-N 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 101150088856 pix gene Proteins 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 210000003720 plasmablast Anatomy 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 210000004043 pneumocyte Anatomy 0.000 description 1
- YJGVMLPVUAXIQN-XVVDYKMHSA-N podophyllotoxin Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-XVVDYKMHSA-N 0.000 description 1
- 229960001237 podophyllotoxin Drugs 0.000 description 1
- YVCVYCSAAZQOJI-UHFFFAOYSA-N podophyllotoxin Natural products COC1=C(O)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YVCVYCSAAZQOJI-UHFFFAOYSA-N 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 108091005493 polytopic transmembrane proteins Proteins 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 235000007686 potassium Nutrition 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 210000001948 pro-b lymphocyte Anatomy 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 229930185346 proliferin Natural products 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 201000001514 prostate carcinoma Diseases 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000006825 purine synthesis Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 239000013608 rAAV vector Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000003439 radiotherapeutic effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 238000013322 recombinant adeno-associated virus production system Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000001533 respiratory mucosa Anatomy 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 230000008207 sensory development Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 230000009131 signaling function Effects 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000012868 site-directed mutagenesis technique Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 108090000586 somatostatin receptor 2 Proteins 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 108091007196 stromelysin Proteins 0.000 description 1
- 230000007847 structural defect Effects 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000000106 sweat gland Anatomy 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 238000010863 targeted diagnosis Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 230000036964 tight binding Effects 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 108091006108 transcriptional coactivators Proteins 0.000 description 1
- 230000037426 transcriptional repression Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 102000025979 tyrosine binding proteins Human genes 0.000 description 1
- 108091009188 tyrosine binding proteins Proteins 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 230000004862 vasculogenesis Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 229960001722 verapamil Drugs 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
- 230000007442 viral DNA synthesis Effects 0.000 description 1
- 230000006656 viral protein synthesis Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to the fields of oncology and molecular biology. More particular the invention relates to the targeting of the Notch3 receptor.
- Lung cancer is the most common cause of cancer-related deaths in the United States.
- the cure rate for patients with lung cancer remains low—15%—and has not changed significantly during the past 30 years (Jermal et al., 2005).
- a better understanding of the signaling pathways important in driving and maintaining the malignant state allows the identification of new therapeutic targets and is thus imperative for continued progress in the treatment of these patients.
- Genes involved in cell fate determination often contribute to tumorigenesis when they are aberrantly expressed.
- the family of Notch receptors is one such family where there are now strong data linking it to cancer pathogenesis.
- Notch3 All four members of the Notch receptor family are known to be dysregulated in the majority of human cancers.
- the inventors were the first to link dysregulation of the Notch3 pathway to human lung cancer (Dang et al., 2000). They demonstrated that Notch3 is highly expressed in 40% of all resected lung cancers and that, in the developing lung, constitutive activation of Notch3 results in inhibition of terminal differentiation. Furthermore, they showed that inhibiting this pathway in human lung tumors results in the loss of the malignant phenotype in vitro and tumor inhibition in xenograft models. This anti-tumor effect is enhanced in the presence of low serum and in combination with an EGFr tyrosine kinase inhibitor. Taken together, these data support an important role for Notch3 and its interaction with the EGF and Ras pathways in lung cancer. However, methods for therapeutic intervention in Notch3 related cancers has not yet been reported.
- an isolated and purified peptide of no more than about 50 residues comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues.
- the peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the peptide may further be comprised in a pharmaceutically acceptable diluent, buffer or excipient.
- a method of inhibiting Notch3 receptor signaling comprising contacting a cell expressing Notch3 with a peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8).
- the cell may be a cancer cell, such as a lung cancer cell and/or an adenocarcinoma.
- More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues.
- the peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the method may further comprise contacting the cell with two or more peptides comprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the method may also further comprises contacting the cancer cell with a second agent that inhibits cancer cell growth, differentiation, metastasis or drug resistance.
- a method of treating a subject having a Notch3-expressing cancer comprising administering to said subject a peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8).
- the subject may a human.
- the cancer cell may be a lung cancer cell and/or an adenocarcinoma.
- More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues.
- the peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the method may further comprise contacting the cell with two or more peptides comprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- the method may also further comprises contacting the cancer cell with a second agent that inhibits cancer cell growth, differentiation, metastasis or drug resistance.
- an isolated and purified antibody that binds to an epitope comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8), as well as methods of using such antibodies to inhibit Notch3 receptor signaling in a cell expressing Notch3.
- a method of treating a subject having a Notch3-expressing cancer comprising administering to said subject an antibody that binds to an epitope comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8).
- Yet another embodiment comprises a pharmaceutical formulation comprising two or more of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10) including three, four, five, six, seven or all of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO
- FIGS. 1A-B Diagram demonstrates the major steps of the canonical Notch pathway.
- FIG. 1A Binding of a DSL ligand triggers proteolysis (S2 and S3) of the Notch receptor and releases the C-terminal NICD fragment.
- FIG. 1B NICD translocates to the nucleus, recruits coactivators, and binds to CSL factors to promote target gene transcription. In the absence of NICD, the CSL factor associates with the corepressor complex and inhibits target gene transcription.
- FIGS. 2A-B Proposed mechanisms of antagonistic and cooperative interactions between Ras and Notch pathways.
- Notch antagonizes Ras signaling by preventing expression of active EGF (1). Notch activation can also directly inhibit Ras activity (2) and induce expression MAPK inhibitors (3).
- FIG. 2B Ras activation in turn can induce expression of Notch inhibitors (4). Paradoxically, Ras activation has also been shown to induce DSL ligand expression (5).
- FIG. 3 Immunohistochemistry with an antibody to extracellular domain of Notch3. Cytoplasmic and membranous staining is observed in squamous cell carcinoma (A) and adenocarcinoma (C) of the lung as compared to aneuroendocrine tumor (B) and normal lung (D). In panel B, slight staining is seen in blood vessels (arrow) within the tumor, consistent with other studies demonstrating normal staining of Notch3 in blood vessels
- FIGS. 4A-D Notch3 alters lung morphology of SP-C-N3IC transgenic mice at E18.5.
- Five ⁇ m-thick lung sections of wild-type littermate controls show that epithelial layer of terminal airways are thin and comprised mostly of type I pneumocytes.
- the terminal lung epithelium of transgenic embryos ( FIGS. 4B , 4 D) demonstrates severe metaplasia, composed mostly of undifferentiated cuboidal cells (arrowheads). No type I pneumocyte was found.
- FIGS. 5A-B Notch inhibition inhibits the tumor phenotype.
- FIG. 5A Inhibition of the Notch3 signaling pathway markedly reduces the size of the colonies formed in soft agar (panel B), compared with vector controls (panel A) in HCC2429 and H460.
- FIG. 5B In serum-starved conditions, the growth of the DN transfectant is severely inhibited in comparison with that of VC. However, with the addition of exogenous growth factors, the growth rate is equal to that of VC.
- FIG. 6 Knocking out Notch3 with siRNA reduces focus formation.
- the pSuper vectors expressing Notch3 siRNA and a mouse Notch3 sequence were transfected into HCC2429, a human lung cancer cell line. The cells were selected with puramycin, then stained with crystal violet after 4 weeks.
- FIGS. 7A-D A ⁇ -secretase inhibitor inhibits tumor cells and Notch3 processing.
- FIG. 7A GSI inhibits HCC2429 cells in a serum-dependent manner. HCC2429 cells are sensitive to GSI, and the sensitivity increases in low serum, similarly to that observed with clones expressing the DN construct. Tumor cells treated with DMSO alone had no change in cell viability.
- FIG. 7B Inhibition of S3 proteolytic processing results in the decrease of Notch3 intracellular domain (N3ICD) and accumulation of S2 product (N3 ⁇ E) after 3 hours.
- FIG. 7C In this experiment, HCC2429 was stably transfected with plasmid vector expressing Notch3 siRNA.
- Control C is the parental HCC2429, whereas siRNA-C clones 5, 6, 8 expressed high level of HCC2429 and siRNA-N3 clones 12, 15, 17 and 20 expressed significantly lower level of Notch3 ( FIG. 7D ). Loss of Notch3 results in no loss in cell survival when treated with MRK003.
- FIGS. 8A-D ⁇ -secretase inhibitor MRK003 demonstrates anti-tumor activity in vivo.
- FIG. 8A Xenografts injected with HCC2429 were treated with MRK003 once the tumors became palpable. After 2 weeks of treatment, the inventors observed about a 50% reduction in tumor size.
- FIG. 8B Loss of activated Notch3 (N3ICD) can be seen in tumor treated with MRK003. Histological examination of resected tumors from xenografts at the end of treatment. Marked necrosis can be seen in the MRK003 treated animal ( FIG. 8D ) as compare to control ( FIG. 8C ).
- FIGS. 9A-C Inhibition of Notch3 increases apoptosis.
- FIG. 9A After 72 hours of exogenous growth factor deprivation, cell lines transfected with DN show a higher percentage of apoptosis as measured by Apo-BrdU analysis.
- FIG. 9B Expression levels of phospho-Akt protein decrease in the Notch3-overexpressing cell line HCC2429 when it is stably transfected with the DN construct, particularly with serum starvation.
- FIG. 9C Transfection with Notch3 SiRNA resulted in loss of Bcl-xL expression and induction of apoptotic product PARP.
- FIGS. 10A-C Notch3 crosstalks with the MAPK pathway.
- FIG. 10A Inhibition of Notch3 signaling in HCC2429 downregulates phospho-p44/42 (ERK?) under serum-starved conditions and after induction with 10% FCS.
- FIG. 10B When the immortalized lung epithelial cell line BEAS-2B was transfected with the DA construct, the inventors observed higher levels of phospho-p44/p42 under serum-starved conditions as well as after serum induction.
- FIG. 10C One mechanism of MAPK modulation includes transcriptional regulation of MKP1 in HCC2429. The DN clones demonstrate significantly higher transcriptional level of MKP1 under serum-starved conditions and at 30 minutes and 1 hour after serum induction, when compared with VC (*).
- FIGS. 11A-B Notch3 modulates the EGF pathway and increases sensitivity to an EGFr inhibitor.
- FIG. 11A In HCC2429, inhibition of the Notch3 pathway increases sensitivity to AG1478 nearly 40-fold.
- IC 50 23.8 ⁇ M
- FIG. 11B A similar observation is made in H460 when the inventors combine AG1478 with L-685,458, a ⁇ -secretase inhibitor, further supporting the hypothesis that EGF cooperates with the Notch pathway in oncogenesis.
- FIGS. 12A-B In HCC2429 lung cancer cell line, Notch inhibitor MRK003 enhances the effect of EGFR inhibitor AG1478 on colony formation in soft agar.
- FIG. 12A Photographs showing that MRK003 not only decreases colony formation, but also enhances the effect of AG1478 on growth.
- FIG. 12B Graph depicts the quantitative decrease in colony formation.
- FIG. 13 Notch3 peptides induce apoptosis.
- the bar graphs here reflect fluorescence counts. Sequences N102: CATAV, N103: CFHGAT, N105: CVSNP, N132: CLNGGS.
- FIG. 14A-B Notch3 peptides induce apoptosis and inhibit Notch3-regulated gene Hey1.
- HCC2429 was treated with Notch3 peptides N16, N17, N102, N103, N132. Induction of apoptosis by peptides is observed as compared to control.
- MRK003-treated cell is used as positive control. After treatment, cells were labeled with annexin V and detected using flow cytometry.
- FIG. 14B Treatment with peptides also reduced transcription of Notch3-dependent gene Hey1 as determined by real-time RT-PCR.
- N17 peptide both demonstrates highest apoptotic activity and best reduction in Hey1 transcription.
- MRK003-treated cells were used as positive control. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N102: CATAV, N103: CFHGAT, N132: CLNGGS.
- FIG. 15 Notch3 Peptides interrupt signaling through binding to ligand Jagged1.
- HEK cells were transfected with Jagged1-HA and treated with Notch3 peptides. The peptides were then immunoprecipitated from cell lysate with streptavidin beads and immunoblotted with anti-HA antibody. No: no input; C: control peptide.
- FIG. 16 Sera from mice immunized with Notch3 recombindant protein inhibit Notch3 activation. Immunoblot demonstrates sera from mice #2, 3, 4, 5, 6 can reduced cleavage of Notch3 ICD (Tx) in Notch3 expressing cell line HCC2429 as compared control (C). Recombinant protein representing EGF-like repeats 21-22 and encompassing sequence CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS was used to immunize AJ and BALB/c mice. HCC2429 was plated in a 6-well plates and treated 1 ⁇ l/ml sera for 24 hr before harvesting.
- FIGS. 17A-B Recombinant Fc-fusion Notch3 proteins inhibit Notch3 activation and induces apoptosis in vitro.
- FIG. 17A Fc-fusion protein comprised for N16-17 and N132 sequences inhibits Notch3 activation. HCC2429 was treated with purified Fc-fusion protein 10 ⁇ g/ml for 24 hrs.
- FIG. 17B Purified recombinant N16-17-Fc protein induces apoptosis as compared to control and Fc control after 40 hrs treatment. Apoptosis was determined by percentage of annexin V positive cells. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N132: CLNGGS.
- Notch receptors are members of an evolutionarily conserved family that is essential for the control of cell fate determination during the development of many multicellular organisms.
- the core components of the Notch pathway are listed in Table 1. Their functions were first discovered in Drosophila melanogaster almost 80 years ago, when a heterozygous deletion was found to result in “notches” at the wing margins. Mutations in Notch genes alter cell fate determination, causing cells destined to become epidermis to instead give rise to neural tissue (reviewed in Artavanis-Tsakonas, 1999).
- Notch signaling is classically divided into two fundamental types: inductive and lateral signaling.
- Induction occurs between two nonequivalent cells, where one cell expresses the receptor and the other expresses the ligand, and through their interaction one cell adopts a different fate.
- lateral signaling occurs between equivalent cells, and through competitive inhibition, adjacent cells are forced to follow a different fate.
- Notch pathway signaling has been studied extensively in lymphogenesis, ondotogenesis, neurogenesis, hair and sensory development (Beatus and Leandahl, 1998; Mitsiadis et al., 1998; Lanford et al., 1999; Robey et al., 1996).
- the expression of Notch receptors is restricted to the vascular systems.
- Notch3 ⁇ / ⁇ mice are fertile and viable, these adult mice exhibit structural defects in the distal arteries and arterial myogenic response, reflecting the lack of proper development in vascular smooth muscle cells (Domenga et al., 2004).
- Notch signaling pathway is important in both vascular development and homeostasis.
- many of the processes involved in embryonic vascular development are mirrored in tumor angiogenesis.
- induction of Notch ligand Jagged1 promotes capillary-like sprout formation in tumor cells (Zeng et al., 2005).
- inhibition of Notch activation by ⁇ -secretase inhibitors also inhibits angiogenesis and tumor proliferation (Paris et al., 2005; Williams et al., 2005).
- Notch1 TAN1
- Notch2 Notch2
- Notch3 the core components of the Notch pathway
- Notch is expressed on cell surfaces as a single-pass, heterodimeric receptor.
- the ligands are also transmembrane proteins of the DSL (Delta/Serrate/LAG-2) family that can be expressed not only on adjacent cells but also on the very same cell expressing the Notch receptors.
- Receptor-ligand interaction triggers proteolysis at the extracellular S2 site near the transmembrane domain and at the S3 site ( FIG. 1 ).
- a TNF- ⁇ converting enzyme (TACE) and a presenillin-1-dependent ⁇ -secretase are believed to be responsible for the proteolytic processing at sites S2 and S3, respectively.
- TACE TNF- ⁇ converting enzyme
- the final cleavage releases the C-terminal, intracellular domain (NICD), which then translocates to the nucleus, recruits coactivators such as mastermind and p300, and binds to CSL (CBF/Suppressor of Hairless/LAG-1) factors.
- CSL proteins in association with corepressors repress target gene transcription.
- Notch signaling causes a switch from transcriptional repression to transcriptional activation of CSL target genes (a review of Notch processing in Mumm and Kopan (2000).
- Notch1 The Notch Signaling Pathway Is Oncogenic. Many key pathways in development play important roles in tumorigenesis when altered. Many key pathways in development play important roles in tumorigenesis when altered. Notch1 was first identified in association with a t(7:9) translocation found in a subset of human T-cell acute lymphoblastoid leukemias (T-ALLs) (Ellisen et al., 1991). While less than 1% of human T-ALLs exhibit the t(7:9), Notch activating mutations have been observed in 50% of human T-ALLs (Weng et al., 2004; Ma et al., 1999). The expression of the constitutively activated intracellular domain (NICD) of Notch1 in bone marrow cells confers an oncogenic phenotype (Pear et al., 1996).
- T-ALLs T-cell acute lymphoblastoid leukemias
- Notch3 has been found to be highly expressed in other tumors, including lung, pancreatic and ovarian carcinoma, using gene expression microarray (Dang et al., 2000; Miyamoto et al., 2003; Lu et al., 2004). Similar studies demonstrate correlations between aberrant Notch ligand/receptor expression and tumor development in various systems (Miyamoto et al., 2003; Santagata et al., 2004; Purow et al., 2005; Callahan and Egan, 2004). The inventors published data demonstrating that inhibition of Notch3 activation using a dominant-negative receptor reduces tumor phenotype (Haruki et al., 2005). Taken together, these observations suggest that the Notch pathway is functionally significant in solid tumors and can serve as a target for therapeutic intervention.
- Notch Crosstalks with the Ras Pathway In both mammals and invertebrates such as Drosophila and C. elegans , Notch receptors signal primarily by the binding to members of the CSL family of transcription factors and related transcription co-activators. However, Notch is known to interact with other pathways including the Wingless/B-catenin and NF- ⁇ B pathways (Johnston and Edgar, 1998; Oswald et al., 1998). One pathway that plays prominently in both development and neoplastic transformation is the EGF/ras/MAPK pathway.
- Notch has been shown in developing organisms to antagonize EGF signaling in cell fate determination through modulation of the MAPK pathway (Faux et al., 2001; Ahmad and Dooley, 1998; Berset et al., 2001). In other cases, however, the Ras and Notch pathways cooperate in promoting certain cell fates (Yoo et al., 2004). As in flies and worms, specific outcomes of EGF and Notch pathways in mammals are context dependent. In mammals, Wang et al. demonstrated that Notch3 induces phosphorylation of ERK1/ERK2 (p44/p42) in vascular smooth muscle cells (Wang et al., 2002).
- FIG. 2 summarizes known potential Notch-ras interactions in both the development and cancer context.
- ⁇ -Secretase Inhibitors Demonstrate Antitumor Effects. Proteolytic processing of Notch receptors following ligand binding is necessary for their activation. The final proteolytic cleavage by the ⁇ -secretase protein complex releases the Notch intracellular domain required for target gene transcription. Thus, pharmacologic intervention that inhibits the activity of any of the proteases can potentially inhibit tumor growth in Notch-dependent cancer. Interestingly, at the same time that presenilins were shown to be essential for Notch signaling, they were discovered as susceptibility loci for Alzheimer's disease (Levitan and Greenwald, 1995).
- Alzheimer's disease The pathogenesis of Alzheimer's disease is believed to be the accumulation of amyloid ⁇ -peptides (A ⁇ ) and formation of amyloid plaques.
- a ⁇ amyloid ⁇ -peptides
- APP ⁇ -amyloid precursor protein
- C99 intermediate fragment
- ⁇ -secretases ⁇ -secretases
- ⁇ -secretase inhibitors block Notch activation and induce apoptosis in multiple cancer cell lines (Qin et al., 2004; Curry et al., 2005; Alves da Costa, 2004). In vivo, these compounds inhibit angiogenesis and tumor growth (Paris et al., 2005). These inhibitors are known to have non-Notch targets, such as erb-4 and CD44, but our data suggest that Notch inhibition may be a component of the observed antitumor effects (Pelletier et al., 2006; Linggi et al., 2006). However, from a practical standpoint, since erb-4 and CD44 are known to be oncogenic, these inhibitors may actually have increased efficacy by virtue of their multiple targets. In fact, a ⁇ -secretase inhibitor by Merck & Co., Inc, is currently in Phase I trials for patients with metastatic or locally advanced breast cancer and for patients with T-cell acute leukemias.
- Notch3 is a 2321 amino acid protein (243659 Da Q9UM47; SEQ ID NO:2) that exists as a heterodimer of a C-terminal fragment (TM) and an N-terminal fragment (EC) which are probably linked by disulfide bonds. It has been shown to iteract with MAML1, MAML2 and MAML3 which act as transcriptional coactivators for NOTCH3. It is localized in the cell membrane and is a single-pass type I membrane protein. Following proteolytical processing, the notch intracellular domain (NICD) causes translocation to the nucleus. Its only known post-translational modification is glycosylation.
- Notch3 functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 to regulate cell-fate determination. Upon ligand activation through the released NICD, it forms a transcriptional activator complex with CBF-1 and activates genes of the enhancer of split locus. As discussed above, it has effects the implementation of differentiation, proliferation and apoptotic programs. It is located at 19p13.2-p13.1.
- Notch3 peptides will comprise molecules of 5 to no more than about 50 residues in length.
- a particular length may be less than 39 residues, less than 35 residues, less than 30 residues, less than 25 residues, less than 20 residues, less than 15 residues, or less than 13, including 5, 6, 7, 8, 9, 10, 11 or 12 residues, and ranges of 5-11 residues, 5-15 residues, 5-20 residues, 5-25 residues, 5-30 residues, 5-35 residues, 5-38 residues, or 5-40 residues.
- the peptides may be generated synthetically or by recombinant techniques, and are purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration), as described in further detail below.
- the peptides may be labeled using various molecules, such as fluorescent, chromogenic or colorimetric agents.
- the peptides may also be linked to other molecules, including other anti-cancer agents.
- the links may be direct or through distinct linker molecules.
- the linker molecules in turn may be subject, in vivo, to cleavage, thereby releasing the agent from the peptide.
- Peptides may also be rendered multimeric by linking to larger, and possibly inert, carrier molecules.
- Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants.
- Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above.
- Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
- Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
- Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
- Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
- amino acids of a protein may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 2 shows the codons that encode particular amino acids.
- the hydropathic index of amino acids may be considered.
- the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
- Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine ( ⁇ 0.4); threonine ( ⁇ 0.7); serine ( ⁇ 0.8); tryptophan ( ⁇ 0.9); tyrosine ( ⁇ 1.3); proline ( ⁇ 1.6); histidine ( ⁇ 3.2); glutamate ( ⁇ 3.5); glutamine ( ⁇ 3.5); aspartate ( ⁇ 3.5); asparagine ( ⁇ 3.5); lysine ( ⁇ 3.9); and arginine ( ⁇ 4.5).
- amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
- substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
- hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( ⁇ 0.4); proline ( ⁇ 0.5 ⁇ 1); alanine ( ⁇ 0.5); histidine * ⁇ 0.5); cysteine ( ⁇ 1.0); methionine ( ⁇ 1.3); valine ( ⁇ 1.5); leucine ( ⁇ 1.8); isoleucine ( ⁇ 1.8); tyrosine ( ⁇ 2.3); phenylalanine ( ⁇ 2.5); tryptophan ( ⁇ 3.4).
- an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
- substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
- amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al., (1993).
- the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
- a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
- a specialized kind of insertional variant is the fusion protein.
- This molecule generally has all or a substantial portion (e.g., an intracellular, transmembrane or extracellular domain) of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide.
- fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
- Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
- Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
- Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
- Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide.
- the term “purified protein or peptide” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
- a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
- purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
- Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
- a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a “-fold purification number.”
- the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
- Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
- High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
- Gel chromatography is a special type of partition chromatography that is based on molecular size.
- the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
- the sole factor determining rate of flow is the size.
- molecules are eluted from the column in decreasing size, so long as the shape is relatively constant.
- Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
- Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
- the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
- Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
- Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fuctose will bind to lectins from lotus.
- the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
- the ligand should be coupled in such a way as to not affect its binding properties.
- the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
- affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
- the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.
- Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference.
- Short peptide sequences, or libraries of overlapping peptides usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
- recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
- the present invention also provides for the use of Notch3 proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either Notch3, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Particular agents are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
- KLH keyhole limpet hemocyannin
- BSA bovine serum albumin
- the present invention also provides, in another embodiment, genes encoding Notch3 or fragments (peptides) thereof.
- a gene for the human Notch3 molecule has been identified.
- the present invention is not limited in scope to this gene, however, as one of ordinary skill in the could readily identify related homologs in various other species (e.g., mouse, rat, rabbit, dog. monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species).
- a “Notch3 gene” may contain a variety of different bases and yet still produce a corresponding polypeptide that is functionally indistinguishable from, and in some cases structurally identical to, the human gene disclosed herein.
- any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid.
- cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate or enhance the function of Notch3.
- a cDNA plus a natural intron or an intron derived from another gene such engineered molecules are sometime referred to as “mini-genes.”
- mini-genes such engineered molecules are sometime referred to as “mini-genes.”
- these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
- cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
- mRNA messenger RNA
- a given Notch3 from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1, above).
- a nucleic acid encoding a Notch3 refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid.
- the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:2.
- the term “as set forth in SEQ ID NO:2” means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:2.
- the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
- Sequences that are essentially the same as those set forth in SEQ ID NO:2 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:2 under standard conditions.
- the DNA segments of the present invention include those encoding biologically functional equivalent Notch3 proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
- functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
- nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules.
- complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:2 under relatively stringent conditions such as those described herein. Such sequences may encode the entire Notch3 protein or functional or non-functional fragments thereof.
- the hybridizing segments may be shorter oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length.
- exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 114 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
- Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
- hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, at temperatures between approximately 20° C. to about 37° C.
- Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 ⁇ M MgCl 2 , at temperatures ranging from approximately 40° C. to about 72° C.
- Formamide and SDS also may be used to alter the hybridization conditions.
- One method of using probes and primers of the present invention is in the search for genes related to Notch3 or, more particularly, homologs of Notch3 from other species.
- the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules.
- stringency of hybridization, and the region of the probe different degrees of homology may be discovered.
- Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
- the technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
- Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
- a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
- the technique typically employs a bacteriophage vector that exists in both a single-stranded and double-stranded form.
- Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
- Double-stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
- site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double-stranded vector which includes within its sequence a DNA sequence encoding the desired protein.
- An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
- This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
- E. coli polymerase I Klenow fragment DNA polymerizing enzymes
- a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
- This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
- sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
- recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
- expression vectors are employed to express the Notch3 polypeptide product, which can then be purified for various uses.
- the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
- expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
- the transcript may be translated into a protein, but it need not be.
- expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
- vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
- a nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
- Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
- plasmids include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
- YACs artificial chromosomes
- expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
- Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
- a “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
- the phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
- a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
- a promoter may be one naturally-associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.”
- an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
- certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
- a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
- Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally-occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
- sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference).
- control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
- promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
- One example is the native Notch3 promoter.
- the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
- the promoter may be heterologous or endogenous.
- Table 3 lists several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof.
- Table 4 provides examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.
- tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
- regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).
- Tumor specific promoters also will find use in the present invention. Some such promoters are set forth in Tables 4 and 5.
- a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
- IRES elements are used to create multigene, or polycistronic, messages.
- IRES elements are able to bypass the ribosome scanning model of 5′-methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
- IRES elements from two members of the picornavirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
- IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.
- each open reading frame is accessible to ribosomes for efficient translation.
- Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, herein incorporated by reference).
- Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
- MCS multiple cloning site
- Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art.
- a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
- “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
- RNA molecules will undergo RNA splicing to remove introns from the primary transcripts.
- Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see Chandler et al., 1997, herein incorporated by reference.)
- the vectors or constructs of the present invention will generally comprise at least one termination signal.
- a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
- the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site.
- RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
- terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.
- the terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.
- Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator.
- the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
- polyadenylation signal In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript.
- the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed.
- Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
- a vector in a host cell may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at which replication is initiated.
- ori origins of replication sites
- ARS autonomously replicating sequence
- cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector.
- markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
- a selectable marker is one that confers a property that allows for selection.
- a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
- An example of a positive selectable marker is a drug resistance marker.
- a drug selection marker aids in the cloning and identification of transformants
- genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
- markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP, whose basis is calorimetric analysis, are also contemplated.
- screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
- viral vectors have led to the development and application of a number of different viral vector systems (Robbins et al., 1998).
- Viral systems are currently being developed for use as vectors for ex vivo and in vivo gene transfer.
- adenovirus, herpes-simplex virus, retrovirus and adeno-associated virus vectors are being evaluated currently for treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, renal disease and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Pat. No. 5,670,488).
- the various viral vectors described below present specific advantages and disadvantages, depending on the particular gene-therapeutic application.
- an adenoviral expression vector is contemplated for the delivery of expression constructs.
- “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein.
- Adenoviruses comprise linear, double-stranded DNA, with a genome ranging from 30 to 35 kb in size (Reddy et al., 1998; Morrison et al., 1997; Chillon et al., 1999).
- An adenovirus expression vector according to the present invention comprises a genetically engineered form of the adenovirus. Advantages of adenoviral gene transfer include the ability to infect a wide variety of cell types, including non-dividing cells, a mid-sized genome, ease of manipulation, high infectivity and the ability to be grown to high titers (Wilson, 1996).
- adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner, without potential genotoxicity associated with other viral vectors.
- Adenoviruses also are structurally stable (Marienfeld et al., 1999) and no genome rearrangement has been detected after extensive amplification (Parks et al., 1997; Bett et al., 1993).
- Salient features of the adenovirus genome are an early region (E1, E2, E3 and E4 genes), an intermediate region (pIX gene, Iva2 gene), a late region (L1, L2, L3, L4 and L5 genes), a major late promoter (MLP), inverted-terminal-repeats (ITRs) and a ⁇ sequence (Zheng, et al., 1999; Robbins et al., 1998; Graham and Prevec, 1995).
- the early genes E1, E2, E3 and E4 are expressed from the virus after infection and encode polypeptides that regulate viral gene expression, cellular gene expression, viral replication, and inhibition of cellular apoptosis.
- the MLP is activated, resulting in the expression of the late (L) genes, encoding polypeptides required for adenovirus encapsidation.
- the intermediate region encodes components of the adenoviral capsid.
- Adenoviral inverted terminal repeats ITRs; 100-200 bp in length
- ITRs are cis elements, and function as origins of replication and are necessary for viral DNA replication.
- the ⁇ sequence is required for the packaging of the adenoviral genome.
- E1 ⁇ the E1 gene
- E2 ⁇ the E2 gene
- E3 and E4 promoters a therapeutic gene or genes can be inserted recombinantly in place of the E1 gene, wherein expression of the therapeutic gene(s) is driven by the E1 promoter or a heterologous promoter.
- the E1 ⁇ , replication-deficient virus is then proliferated in a “helper” cell line that provides the E1 polypeptides in trans (e.g., the human embryonic kidney cell line 293).
- the present invention it may be convenient to introduce the transforming construct at the position from which the E1-coding sequences have been removed.
- the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
- the E3 region, portions of the E4 region or both may be deleted, wherein a heterologous nucleic acid sequence under the control of a promoter operable in eukaryotic cells is inserted into the adenovirus genome for use in gene transfer (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210, each specifically incorporated herein by reference).
- adenovirus based vectors offer several unique advantages over other vector systems, they often are limited by vector immunogenicity, size constraints for insertion of recombinant genes and low levels of replication.
- the preparation of a recombinant adenovirus vector deleted of all open reading frames, comprising a full length dystrophin gene and the terminal repeats required for replication offers some potentially promising advantages to the above mentioned adenoviral shortcomings.
- the vector was grown to high titer with a helper virus in 293 cells and was capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Helper-dependent viral vectors are discussed below.
- a major concern in using adenoviral vectors is the generation of a replication-competent virus during vector production in a packaging cell line or during gene therapy treatment of an individual.
- the generation of a replication-competent virus could pose serious threat of an unintended viral infection and pathological consequences for the patient.
- Armentano et al. (1990) describe the preparation of a replication-defective adenovirus vector, claimed to eliminate the potential for the inadvertent generation of a replication-competent adenovirus (U.S. Pat. No. 5,824,544, specifically incorporated herein by reference).
- the replication-defective adenovirus method comprises a deleted E1 region and a relocated protein IX gene, wherein the vector expresses a heterologous, mammalian gene.
- the adenovirus may be of any of the 42 different known serotypes and/or subgroups A-F.
- Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
- the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region.
- Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210; U.S. Pat. No. 5,824,544).
- This group of viruses can be obtained in high titers, e.g., 10 9 to 10 11 plaque-forming units per ml, and they are highly infective.
- the life cycle of adenovirus does not require integration into the host cell genome.
- adenoviral gene delivery-based gene therapies are being developed for liver diseases (Han et al., 1999), psychiatric diseases (Lesch, 1999), neurological diseases (Smith, 1998; Hermens and Verhaagen, 1998), coronary diseases (Feldman et al., 1996), muscular diseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998) and various cancers such as colorectal (Fujiwara and Tanaka, 1998; Dorai et al., 1999), pancreatic, bladder (Irie et al., 1999), head and neck (Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra et al., 1999) and ovarian (V
- Retroviral Vectors In certain embodiments of the invention, the use of retroviruses for gene delivery are contemplated. Retroviruses are RNA viruses comprising an RNA genome. When a host cell is infected by a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated into the chromosomal DNA of infected cells. This integrated DNA intermediate is referred to as a provirus.
- retroviruses A particular advantage of retroviruses is that they can stably infect dividing cells with a gene of interest (e.g., a therapeutic gene) by integrating into the host DNA, without expressing immunogenic viral proteins. Theoretically, the integrated retroviral vector will be maintained for the life of the infected host cell, expressing the gene of interest.
- the retroviral genome and the proviral DNA have three genes: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences.
- the gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase) and the env gene encodes viral envelope glycoproteins.
- the 5′ and 3′ LTRs serve to promote transcription and polyadenylation of the virion RNAs.
- the LTR contains all other cis-acting sequences necessary for viral replication.
- a recombinant retrovirus of the present invention may be genetically modified in such a way that some of the structural, infectious genes of the native virus have been removed and replaced instead with a nucleic acid sequence to be delivered to a target cell (U.S. Pat. No. 5,858,744; U.S. Pat. No. 5,739,018, each incorporated herein by reference).
- the virus injects its nucleic acid into the cell and the retrovirus genetic material can integrate into the host cell genome.
- the transferred retrovirus genetic material is then transcribed and translated into proteins within the host cell.
- the generation of a replication-competent retrovirus during vector production or during therapy is a major concern.
- Retroviral vectors suitable for use in the present invention are generally defective retroviral vectors that are capable of infecting the target cell, reverse transcribing their RNA genomes, and integrating the reverse transcribed DNA into the target cell genome, but are incapable of replicating within the target cell to produce infectious retroviral particles (e.g., the retroviral genome transferred into the target cell is defective in gag, the gene encoding virion structural proteins, and/or in pol, the gene encoding reverse transcriptase).
- infectious retroviral particles e.g., the retroviral genome transferred into the target cell is defective in gag, the gene encoding virion structural proteins, and/or in pol, the gene encoding reverse transcriptase.
- transcription of the provirus and assembly into infectious virus occurs in the presence of an appropriate helper virus or in a cell line containing appropriate sequences enabling encapsidation without coincident production of a contaminating helper virus.
- retroviruses The growth and maintenance of retroviruses is known in the art (U.S. Pat. No. 5,955,331; U.S. Pat. No. 5,888,502, each specifically incorporated herein by reference).
- Nolan et al. describe the production of stable high titre, helper-free retrovirus comprising a heterologous gene (U.S. Pat. No. 5,830,725, specifically incorporated herein by reference).
- retroviral gene delivery includes a requirement for ongoing cell division for stable infection and a coding capacity that prevents the delivery of large genes.
- genes via retroviruses are currently being assessed for the treatment of various disorders such as inflammatory disease (Moldawer et al., 1999), AIDS (Amado and Chen, 1999; Engel and Kohn, 1999), cancer (Clay et al., 1999), cerebrovascular disease (Weihl et al., 1999) and hemophilia (Kay, 1998).
- Herpesviral Vectors Herpes simplex virus (HSV) type I and type II contain a double-stranded, linear DNA genome of approximately 150 kb, encoding 70-80 genes. Wild type HSV are able to infect cells lytically and to establish latency in certain cell types (e.g., neurons).
- HSV Herpes simplex virus
- HSV Similar to adenovirus, HSV also can infect a variety of cell types including muscle (Yeung et al., 1999), ear (Derby et al., 1999), eye (Kaufman et al., 1999), tumors (Yoon et al., 1999; Howard et al., 1999), lung (Kohut et al., 1998), neuronal (Gamido et al., 1999; Lachmann and Efstathiou, 1999), liver (Miyatake et al., 1999; Kooby et al., 1999) and pancreatic islets (Rabinovitch et al., 1999).
- HSV viral genes are transcribed by cellular RNA polymerase II and are temporally regulated, resulting in the transcription and subsequent synthesis of gene products in roughly three discernable phases or kinetic classes. These phases of genes are referred to as the Immediate Early (IE) or alpha genes, Early (E) or beta genes and Late (L) or gamma genes. Immediately following the arrival of the genome of a virus in the nucleus of a newly infected cell, the IE genes are transcribed. The efficient expression of these genes does not require prior viral protein synthesis. The products of IE genes are required to activate transcription and regulate the remainder of the viral genome.
- IE Immediate Early
- E Early
- L Late
- ICP4 Infected Cell Polypeptide 4
- viruses deleted of ICP4 Phenotypic studies of HSV viruses deleted of ICP4 indicate that such viruses will be potentially useful for gene transfer purposes (Krisky et al., 1998a).
- One property of viruses deleted for ICP4 that makes them desirable for gene transfer is that they only express the five other IE genes: ICP0, ICP6, ICP27, ICP22 and ICP47 (DeLuca et al., 1985), without the expression of viral genes encoding proteins that direct viral DNA synthesis, as well as the structural proteins of the virus. This property is desirable for minimizing possible deleterious effects on host cell metabolism or an immune response following gene transfer.
- Further deletion of IE genes ICP22 and ICP27, in addition to ICP4, substantially improve reduction of HSV cytotoxicity and prevented early and late viral gene expression (Krisky et al., 1998b).
- HSV HSV in gene transfer
- diseases such as Parkinson's (Yamada et al., 1999), retinoblastoma (Hayashi et al., 1999), intracerebral and intradermal tumors (Moriuchi et al., 1998), B-cell malignancies (Suzuki et al., 1998), ovarian cancer (Wang et al., 1998) and Duchenne muscular dystrophy (Huard et al., 1997).
- Adeno-associated virus a member of the parvovirus family, is a human virus that is increasingly being used for gene delivery therapeutics.
- AAV has several advantageous features not found in other viral systems. First, AAV can infect a wide range of host cells, including non-dividing cells. Second, AAV can infect cells from different species. Third, AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. For example, it is estimated that 80-85% of the human population has been exposed to AAV. Finally, AAV is stable at a wide range of physical and chemical conditions which lends itself to production, storage and transportation requirements.
- the AAV genome is a linear, single-stranded DNA molecule containing 4681 nucleotides.
- the AAV genome generally comprises an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs) of approximately 145 bp in length.
- ITRs inverted terminal repeats
- the ITRs have multiple functions, including origins of DNA replication, and as packaging signals for the viral genome.
- the internal non-repeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes.
- the rep and cap genes code for viral proteins that allow the virus to replicate and package the viral genome into a virion.
- a family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight.
- the AAV cap region encodes at least three proteins, VP1, VP2, and VP3.
- AAV is a helper-dependent virus requiring co-infection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia) in order to form AAV virions.
- a helper virus e.g., adenovirus, herpesvirus or vaccinia
- AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced.
- Subsequent infection by a helper virus “rescues” the integrated genome, allowing it to replicate and package its genome into infectious AAV virions.
- the helper virus must be of the same species as the host cell (e.g., human AAV will replicate in canine cells co-infected with a canine adenovirus).
- AAV has been engineered to deliver genes of interest by deleting the internal non-repeating portion of the AAV genome and inserting a heterologous gene between the ITRs.
- the heterologous gene may be functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in target cells.
- a suitable producer cell line is transfected with a rAAV vector containing a heterologous gene.
- the producer cell is concurrently transfected with a second plasmid harboring the AAV rep and cap genes under the control of their respective endogenous promoters or heterologous promoters.
- the producer cell is infected with a helper virus.
- the heterologous gene is replicated and packaged as though it were a wild-type AAV genome.
- target cells are infected with the resulting rAAV virions, the heterologous gene enters and is expressed in the target cells. Because the target cells lack the rep and cap genes and the adenovirus helper genes, the rAAV cannot further replicate, package or form wild-type AAV.
- helper virus presents a number of problems.
- the contaminating infectious adenovirus can be inactivated by heat treatment (56° C. for 1 hour). Heat treatment, however, results in approximately a 50% drop in the titer of functional rAAV virions.
- Second, varying amounts of adenovirus proteins are present in these preparations. For example, approximately 50% or greater of the total protein obtained in such rAAV virion preparations is free adenovirus fiber protein. If not completely removed, these adenovirus proteins have the potential of eliciting an immune response from the patient.
- helper virus particles in rAAV virion producing cells diverts large amounts of host cellular resources away from rAAV virion production, potentially resulting in lower rAAV virion yields.
- Lentiviral Vectors are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection.
- Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV.
- Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
- Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
- the lentiviral genome and the proviral DNA have the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences.
- the gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins;
- the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), a protease and an integrase; and the env gene encodes viral envelope glycoproteins.
- the 5′ and 3′ LTR's serve to promote transcription and polyadenylation of the virion RNA's.
- the LTR contains all other cis-acting sequences necessary for viral replication.
- Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx.
- Adjacent to the 5′ LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.
- Lentiviral vectors are known in the art, see Naldini et al., (1996); Zufferey et al., (1997); U.S. Pat. Nos. 6,013,516; and 5,994,136.
- the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell.
- the gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
- Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
- This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell.
- Introducing a vector providing a heterologous gene, such as the STAT-1 ⁇ gene in this invention, into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest.
- the env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.
- a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
- the vector providing the viral env nucleic acid sequence is associated operably with regulatory sequences, e.g., a promoter or enhancer.
- the regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences.
- the heterologous or foreign nucleic acid sequence is linked operably to a regulatory nucleic acid sequence.
- the heterologous sequence is linked to a promoter, resulting in a chimeric gene.
- the heterologous nucleic acid sequence may also be under control of either the viral LTR promoter-enhancer signals or of an internal promoter, and retained signals within the retroviral LTR can still bring about efficient expression of the transgene.
- Marker genes may be utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of a marker gene ensures the selection and growth of only those host cells which express the inserts.
- Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate, etc., and cell surface markers.
- the vectors are introduced via transfection or infection into the packaging cell line.
- the packaging cell line produces viral particles that contain the vector genome. Methods for transfection or infection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art.
- the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.
- the selectable marker gene can be linked physically to the packaging genes in the construct.
- Lentiviral transfer vectors Naldini et al. (1996) have been used to infect human cells growth-arrested in vitro and to transduce neurons after direct injection into the brain of adult rats.
- the vector was efficient at transferring marker genes in vivo into the neurons and long term expression in the absence of detectable pathology was achieved.
- Animals analyzed ten months after a single injection of the vector showed no decrease in the average level of transgene expression and no sign of tissue pathology or immune reaction (Blomer et al., 1997).
- one may graft or transplant cells infected with the recombinant lentivirus ex vivo, or infect cells in vivo.
- viral vectors for gene delivery.
- Other viral vectors such as poxvirus; e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alpha virus; e.g., Sindbis virus, Semliki forest virus (Lundstrom, 1999), reovirus (Coffey et al., 1998) and influenza A virus (Neumann et al., 1999) are contemplated for use in the present invention and may be selected according to the requisite properties of the target system.
- poxvirus e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alpha virus; e.g., Sindbis virus, Semliki forest virus (Lundstrom, 1999), reovirus (Coffey et al., 1998) and influenza A virus (Neumann et al., 1999) are contemplated for use in the present invention and may be selected according to the requisite properties of
- vaccinia viral vectors are contemplated for use in the present invention.
- Vaccinia virus is a particularly useful eukaryotic viral vector system for expressing heterologous genes. For example, when recombinant vaccinia virus is properly engineered, the proteins are synthesized, processed and transported to the plasma membrane.
- Vaccinia viruses as gene delivery vectors have recently been demonstrated to transfer genes to human tumor cells, e.g., EMAP-II (Gnant et al., 1999), inner ear (Derby et al., 1999), glioma cells, e.g., p53 (Timiryasova et al., 1999) and various mammalian cells, e.g., P-450 (U.S. Pat. No. 5,506,138).
- EMAP-II Gnant et al., 1999
- inner ear Deby et al., 1999
- glioma cells e.g., p53 (Timiryasova et al., 1999)
- various mammalian cells e.g., P-450 (U.S. Pat. No. 5,506,138).
- the preparation, growth and manipulation of vaccinia viruses are described in U.S. Pat. No. 5,849,304 and U.S. Pat. No
- Sindbis viral vectors are contemplated for use in gene delivery.
- Sindbis virus is a species of the alphavirus genus (Garoff and Li, 1998) which includes such important pathogens as Venezuelan, Western and Eastern equine encephalitis viruses (Sawai et al., 1999; Mastrangelo et al., 1999).
- Sindbis virus infects a variety of avian, mammalian, reptilian, and amphibian cells.
- the genome of Sindbis virus consists of a single molecule of single-stranded RNA, 11,703 nucleotides in length.
- the genomic RNA is infectious, is capped at the 5′ terminus and polyadenylated at the 3′ terminus, and serves as mRNA.
- Translation of a vaccinia virus 26S mRNA produces a polyprotein that is cleaved co- and post-translationally by a combination of viral and presumably host-encoded proteases to give the three virus structural proteins, a capsid protein (C) and the two envelope glycoproteins (E1 and PE2, precursors of the virion E2).
- Sindbis virus Three features suggest that it would be a useful vector for the expression of heterologous genes. First, its wide host range, both in nature and in the laboratory. Second, gene expression occurs in the cytoplasm of the host cell and is rapid and efficient. Third, temperature-sensitive mutations in RNA synthesis are available that may be used to modulate the expression of heterologous coding sequences by simply shifting cultures to the non-permissive temperature at various time after infection. The growth and maintenance of Sindbis virus is known in the art (U.S. Pat. No. 5,217,879, specifically incorporated herein by reference).
- Chimeric Viral Vectors Chimeric or hybrid viral vectors are being developed for use in therapeutic gene delivery and are contemplated for use in the present invention. Chimeric poxyiral/retroviral vectors (Holzer et al., 1999), adenoviral/retroviral vectors (Feng et al., 1997; Bilbao et al., 1999; Caplen et al., 1999) and adenoviral/adeno-associated viral vectors (Fisher et al., 1996; U.S. Pat. No. 5,871,982) have been described.
- Wilson et al. provide a chimeric vector construct which comprises a portion of an adenovirus, AAV 5′ and 3′ ITR sequences and a selected transgene, described below (U.S. Pat. No. 5,871,983, specifically incorporate herein by reference).
- the adenovirus/AAV chimeric virus uses adenovirus nucleic acid sequences as a shuttle to deliver a recombinant AAV/transgene genome to a target cell.
- the adenovirus nucleic acid sequences employed in the hybrid vector can range from a minimum sequence amount, which requires the use of a helper virus to produce the hybrid virus particle, to only selected deletions of adenovirus genes, which deleted gene products can be supplied in the hybrid viral production process by a selected packaging cell.
- the adenovirus nucleic acid sequences employed in the pAdA shuttle vector are adenovirus genomic sequences from which all viral genes are deleted and which contain only those adenovirus sequences required for packaging adenoviral genomic DNA into a preformed capsid head. More specifically, the adenovirus sequences employed are the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences of an adenovirus (which function as origins of replication) and the native 5′ packaging/enhancer domain, that contains sequences necessary for packaging linear Ad genomes and enhancer elements for the E1 promoter.
- ITR inverted terminal repeat
- the adenovirus sequences may be modified to contain desired deletions, substitutions, or mutations, provided that the desired function is not eliminated.
- the AAV sequences useful in the above chimeric vector are the viral sequences from which the rep and cap polypeptide encoding sequences are deleted. More specifically, the AAV sequences employed are the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences. These chimeras are characterized by high titer transgene delivery to a host cell and the ability to stably integrate the transgene into the host cell chromosome (U.S. Pat. No. 5,871,983, specifically incorporate herein by reference). In the hybrid vector construct, the AAV sequences are flanked by the selected adenovirus sequences discussed above. The 5′ and 3′ AAV ITR sequences themselves flank a selected transgene sequence and associated regulatory elements, described below.
- ITR inverted terminal repeat
- the sequence formed by the transgene and flanking 5′ and 3′ AAV sequences may be inserted at any deletion site in the adenovirus sequences of the vector.
- the AAV sequences are desirably inserted at the site of the deleted E1a/E1b genes of the adenovirus.
- the AAV sequences may be inserted at an E3 deletion, E2a deletion, and so on. If only the adenovirus 5′ ITR/packaging sequences and 3′ ITR sequences are used in the hybrid virus, the AAV sequences are inserted between them.
- the transgene sequence of the vector and recombinant virus can be a gene, a nucleic acid sequence or reverse transcript thereof, heterologous to the adenovirus sequence, which encodes a protein, polypeptide or peptide fragment of interest.
- the transgene is operatively linked to regulatory components in a manner which permits transgene transcription.
- the composition of the transgene sequence will depend upon the use to which the resulting hybrid vector will be put.
- one type of transgene sequence includes a therapeutic gene which expresses a desired gene product in a host cell.
- These therapeutic genes or nucleic acid sequences typically encode products for administration and expression in a patient in vivo or ex vivo to replace or correct an inherited or non-inherited genetic defect or treat an epigenetic disorder or disease.
- a nucleic acid e.g., DNA
- Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos.
- a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, either subcutaneously, intradermally, intramuscularly, intervenously or intraperitoneally.
- injections i.e., a needle injection
- Methods of injection of vaccines are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution).
- Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985).
- Electroporation In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384,253, incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.
- friable tissues such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly.
- pectolyases pectolyases
- mechanically wounding in a controlled manner.
- pectolyases pectolyases
- One also may employ protoplasts for electroporation transformation of plant cells (Bates, 1994; Lazzeri, 1995).
- protoplasts for electroporation transformation of plant cells
- the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application No. WO 9217598, incorporated herein by reference.
- Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato (Tsukada, 1989).
- a nucleic acid is introduced to the cells using calcium phosphate precipitation.
- Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique.
- mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).
- DEAE-Dextran In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
- Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonic loading.
- LTK ⁇ fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987).
- a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome.
- Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).
- Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987).
- the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).
- a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989).
- a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991).
- HMG-1 nuclear non-histone chromosomal proteins
- a liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
- a delivery vehicle may comprise a ligand and a liposome.
- a nucleic acid may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.
- Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached.
- Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated herein by reference).
- a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.
- a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome.
- the nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane.
- the liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell.
- Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.
- EGF epidermal growth factor
- the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
- lipids or glycoproteins that direct cell-specific binding.
- lactosyl-ceramide, a galactose-terminal asialganglioside have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner.
- prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
- the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name M AX B AC ® 2.0 from I NVITROGEN ® and B AC P ACK TM B ACULOVIRUS E XPRESSION S YSTEM F ROM C LONTECH®.
- expression systems include S TRATAGENE ®'S C OMPLETE C ONTROL TM Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
- I NVITROGEN ® which carries the T-R EX TM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
- I NVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica .
- a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
- Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented.
- One embodiment of the foregoing involves the use of gene transfer to immortalize cells for the production of proteins.
- the gene for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions.
- the gene for virtually any polypeptide may be employed in this manner.
- the generation of recombinant expression vectors, and the elements included therein, are discussed above.
- the protein to be produced may be an endogenous protein normally synthesized by the cell in question.
- Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells.
- a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
- a number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk ⁇ , hgprt ⁇ or aprt ⁇ cells, respectively.
- anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
- the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
- “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
- a host cell can, and has been, used as a recipient for vectors.
- a host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
- a transformed cell includes the primary subject cell and its progeny.
- Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences.
- Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).
- ATCC American Type Culture Collection
- An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
- a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
- Bacterial cells used as host cells for vector replication and/or expression include DH5 ⁇ , JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACKTM Gold Cells (STRATAGENE®, La Jolla).
- bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
- eukaryotic host cells for replication and/or expression of a vector examples include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
- Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
- control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
- One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
- Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).
- Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
- suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
- the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture.
- the gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor.
- the main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1.
- the airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
- the antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation.
- Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
- For the isolation of membrane proteins cells must be solubilized into detergent micelles.
- Non-ionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
- Antibodies are and their uses are discussed further, below.
- the present invention contemplates an antibody that is immunoreactive with a Notch3 molecule of the present invention, or any portion thereof.
- the invention contemplates using peptides having the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, optionally linked together or linked to a carrier molecule such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988).
- a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide, of the present invention and collecting antisera from that immunized animal.
- an immunogen comprising a polypeptide, of the present invention
- a wide range of animal species can be used for the production of antisera.
- an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
- Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
- a composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention.
- Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
- the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to Notch3-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular Notch3 of different species may be utilized in other useful applications
- both polyclonal and monoclonal antibodies against Notch3 may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other Notch3. They may also be used in inhibition studies to analyze the effects of Notch3 related peptides in cells or animals. Anti-Notch3 antibodies will also be useful in immunolocalization studies to analyze the distribution of Notch3 during various cellular events, for example, to determine the cellular or tissue-specific distribution of Notch3 polypeptides under different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant Notch3, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
- a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
- exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
- Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
- the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
- adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis ), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
- the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
- a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
- the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
- the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
- MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference.
- this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified Notch3 protein, polypeptide or peptide or cell expressing high levels of Notch3.
- the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
- Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible.
- the use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
- somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol.
- B-cells B-lymphocytes
- These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
- a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
- a spleen from an immunized mouse contains approximately 5 ⁇ 10 7 to 2 ⁇ 10 8 lymphocytes.
- the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
- Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
- any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
- the immunized animal is a mouse
- P3-X63/Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.
- Fusion procedures usually produce viable hybrids at low frequencies, around 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
- the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
- Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
- This culturing provides a population of hybridomas from which specific hybridomas are selected.
- selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
- the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
- the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
- the cell lines may be exploited for mAb production in two basic ways.
- a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
- the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
- the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
- the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
- mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
- the present invention also involves, in another embodiment, the treatment of cancer.
- the types of cancer that may be treated, according to the present invention is limited only by the involvement of Notch3. By involvement, it is not even a requirement that Notch3 be mutated or abnormal—the overexpression of this tumor suppressor may actually overcome other lesions within the cell.
- Notch3 therapy including cancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue.
- the tumor cell be killed or induced to undergo normal cell death or “apoptosis.” Rather, to accomplish a meaningful treatment, all that is required is that the tumor growth be slowed to some degree. It may be that the tumor growth is completely blocked, however, or that some tumor regression is achieved. Clinical terminology such as “remission” and “reduction of tumor” burden also are contemplated given their normal usage.
- Notch3 polypeptide fragments, synthetic peptides, mimetics or other analogs thereof.
- the protein/peptide may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer.
- Formulations would be selected based on the route of administration and purpose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
- Antibodies will be administered according to standard protocols for passive immunotherapy. Administration protocols would generally involve intratumoral, local or regional (to the tumor) administration, as well as systemic administration.
- the antibody reagent may be altered, such that it will have one or more improved properties.
- the antibody may be recombinant, i.e., an antibody gene cloned into an expression cassette which is then introduced into a cell in which the antibody gene was not initially created.
- the antibody may be single chain, a fragment (Fab, Fv, Vh, ScFv), chimeric or humanized.
- HS-tk herpes simplex-thymidine kinase
- compositions of the present invention To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one would generally contact a “target” cell with a Notch3 peptide or antibody and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with a Notch3 peptide or antibody and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
- the Notch3 peptide or antibody therapy treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
- the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and a Notch3 peptide or antibody would still be able to exert an advantageously combined effect on the cell.
- Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell.
- Such agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
- Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.
- the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
- the use of cisplatin in combination with a Notch3 expression construct is particularly preferred as this compound.
- the tumor cells In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, ⁇ -rays or even microwaves.
- the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.
- the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a Notch3 expression construct, as described above.
- Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
- chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m 2 at 21 day intervals for adriamycin, to 35-50 mg/m 2 for etoposide intravenously or double the intravenous dose orally.
- ⁇ -rays X-rays
- X-rays X-rays
- UV-irradiation UV-irradiation
- Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens.
- Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
- Notch3 expression constructs to patients with cancer will be a very efficient method for treating the clinical disease.
- the chemo- or radiotherapy may be directed to a particular, affected region of the subjects body.
- systemic delivery of expression construct and/or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
- Notch3 therapies with chemo- and radiotherapies
- combination with other gene therapies will be advantageous.
- targeting of Notch3 and p53 mutations at the same time may produce an improved anti-cancer treatment.
- Any other tumor-related gene conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.
- any of the foregoing therapies may prove useful by themselves in treating a Notch3.
- reference to chemotherapeutics and non-Notch3 gene therapy in combination should also be read as a contemplation that these approaches may be employed separately.
- compositions a Notch3 peptide, or antibody—in a form appropriate for the intended application.
- this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
- Aqueous compositions of the present invention comprise an effective amount of the Notch3 peptide or antibody to cells/a subject, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. Of particular interest is direct intratumoral administration, perfusion of a tumor, or administration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed.
- the active compounds may also be administered parenterally or intraperitoneally.
- Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- a coating such as lecithin
- surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- the Notch3 peptides or antibodies of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
- a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
- the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
- the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
- the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
- compositions of the present invention may be formulated in a neutral or salt form.
- Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
- solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
- the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
- sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
- IHC immunohistochemistry
- the inventors used tumor tissue arrays produced as part of the Vanderbilt SPORE initiative.
- the frequency of Notch3 overexpression in lung tumors is higher than the frequency of HER2/neu (16%) and k-Ras mutations (16%), and comparable to EGFr (13-80%) (Hirsch et al., 2002; Rodenhuis et al., 1997; Meert et al., 2002).
- No information is available with regard to the frequency of overexpression of other members of the Notch family or their receptors in human lung carcinomas.
- the lack of high quality antibodies to the Notch receptors, Jagged1, and Delta-like-1, -3 and -4 makes it difficult to determine their frequency in fresh tumor tissues.
- the Notch pathway crosstalks with the EGF pathway in cell fate determination during development.
- the inventors thus examined the correlation between Notch3 and EGFr expression.
- the frequency of EGFr expression in our tumor tissue arrays was 81%. 19% of EGFr positive tumors are Notch3 negative, whereas 43% of EGFr positive tumors are also Notch3 positive (p ⁇ 0.0001 using Pearson correlation coefficient) (Haruki et al., 2005). This highly statistical association suggests that the Notch and EGF pathways are cooperative in lung tumorigenesis.
- Ectopic Notch3 Expression Inhibits Terminal Differentiation in Developing Lungs of Transgenic Mice.
- the inventors studied the effect of activated Notch3 using a lung-specific, human SP-C promoter (Glasser et al., 1991; Lardelli et al., 1996).
- the constitutively-activate Notch3 allowed the inventors to assess the effect of ectopic expression of Notch3 without depending on ligand expression in the epithelium, since Jagged1 expression becomes restricted to endothelium as the lung matures (Taichman et al., 2002).
- constitutive expression of Notch3 also better mimics the many dysregulated pathways observed in cancer.
- the inventors observed perinatal lethality, and thus no surviving animals expresses the N3IC transgene.
- the transgenic mice were sacrificed prior to birth, at E18.5. Of the 182 embryos at E18.5 gestation collected from 32 pregnant mothers, 10 were transgenic, as determined by PCR and Southern blot analysis. The inventors observed altered lung morphogenesis, altered terminal sac morphology, and abnormally abundant mesenchyme, and no type I pneumocytes when compared with control mice ( FIGS. 4A-D ).
- this transgenic model provides evidence that dysregulation of Notch3 signaling in the developing lung affects morphogenesis and terminal differentiation of the lung epithelium.
- Prenatal activation of Notch3 in the peripheral epithelium of the lung in our SP-C-N3IC mouse model leads to nonviable newborn pups, making it impossible to evaluate tumor progression.
- An inducible transgenic model that allows activation of Notch3 signaling in the postnatal period will be more effectively recapitulate the somatic activation of potential oncogenes observed in human lung cancer. This approach is part of the proposal in our R01 funding. Regardless, the ectopic expression of Notch3 appears to inhibit terminal differentiation of type I pneumocytes and results in metaplasia of the immature respiratory epithelium, supporting a potential role of Notch3 in lung cancer formation.
- Notch3 Inhibition Inhibits the Tumor Phenotype.
- the inventors used the approach of uncoupling its ligand binding and signaling functions (Rebay et al., 1993).
- the inventors created a dominant-negative (DN) construct.
- DN dominant-negative
- One characteristic feature of malignant transformation is the ability of tumor cells to proliferate in the absence of adhesion, as measured by the soft-agar assay for colony formation.
- the inventors demonstrated that inhibition of Notch3 signaling by the DN construct dramatically decreases the ability of HCC2429 and H460 to form colonies in soft agar ( FIG. 5A ).
- a ⁇ -Secretase Inhibitor Reduces Proliferation in Lung Cancer Cells.
- Proteolytic processing of Notch receptors is required for activation.
- three proteolytic cleavage sites are involved in enabling Notch signaling.
- the membrane-associated Notch fragment is cleaved within its transmembrane domain by a ⁇ -secretase-containing protein complex.
- Mammalian presenilins PS-1 and PS-2 are polytopic transmembrane proteins that appear to function as aspartyl proteases within a multiprotein, ⁇ -secretase complex (Wolfe and Kopan, 2004). Presenilins provide the active site of the proteolytic activity.
- GSI Gamma Secretase Inhibitor
- DMSO DMSO
- the inventors also observed inhibition of proliferation in other lung cancer cell lines expressing Notch3 (data not shown). Biochemically, treatment with these inhibitors also resulted in the loss of activated Notch3, as measured by the levels of the N3ICD ( FIG. 7B ). Stable expression of Notch3 siRNA also leads to loss of sensitivity to ⁇ -secretase inhibition ( FIGS. 7C , 7 D).
- mice were treated subcutaneous xenografts with MRK003, a ⁇ -secretase inhibitor from Merck, Inc. & Co. Based on the manufacturer's recommended dose, the mice were treated with 100 mg/kg orally for 3 consecutives days per week once the tumors were palpable. At the end of a 2-week treatment, the animals were then euthanized, and the tumors were harvested. A reduction of tumor size was seen in animals treated with the inhibitor, with a concomitant reduction in activated Notch3 (N3ICD) ( FIGS. 8A-D ).
- N3ICD activated Notch3
- Notch3 Inhibition Induces Apoptosis.
- One hallmark of cancer is resistance to apoptosis.
- Genetic and pharmacologic inhibition of the Notch pathway in other tumors has been shown to induce apoptosis (Qin et al., 2004; Curry et al., 2005; Shelly et al., 1999).
- the inventors examined whether inhibiting the Notch3 pathway can induce apoptosis.
- FIG. 9B shows that the level of phosphorylated Akt does indeed decrease when the DN Notch3 construct is used.
- the inventors also demonstrated that inhibition of Notch3 using siRNA results in the down-regulation of Bcl-xL (24 and 48 hours) and enhances the upregulation of cleaved PARP (48 hours), suggesting that Notch3 plays and important role in tumor survival ( FIG. 9C ).
- Notch3 Crosstalks with the MAPK Pathway.
- Notch receptors signal primarily by binding to CBF-1 and related transcription co-activators.
- the Notch and EGF/MAPK pathways are known to interact in developing vertebrates and invertebrates.
- the MAPK pathway and in particular the ERK subfamily, also plays a prominent role in cellular response to growth factors, and is often altered in cancer.
- the inventors examined whether Notch3 alters ERK signaling in lung cancer cells. Using the DN receptor, they showed that inhibiting Notch3 downregulates MAPK, and conversely, that MAPK is upregulated when the cells were transfected with activated Notch3 intracellular domain ( FIGS. 10A , 10 B).
- the HCC2429 clone expressing the dominant-negative construct shows that p44/p42 phosphorylation was attenuated significantly compared to vector control ( FIG. 10A ).
- Prolonged exposure to growth factors and activation of the p44/p42 cascade can induce the expression of MAPK phosphatases-1 and -2 (MKP-1/-2) as part of a negative feedback loop and result in the down-regulation of the MAPK pathway (Traverse et al., 1992; Plows et al., 2002; Haneda et al., 1999; Brondello et al., 1997).
- Notch3 may have a role in suppressing MKP expression (Sivaraman et al., 1997; Magi-galluzzi et al., 1997; Barry et al., 2001). Recent data demonstrate that one mechanism by which Notch antagonizes EGF in developing C. elegans is the upregulation of LIP-1, a homolog of mammalian MAPK phosphatases (Berset et al., 2001). Since Drosophila and C.
- Notch3 might suppress MAPK phosphatase expression in cancer cells.
- the inventors quantitated the level of MKP-1 using real-time PCR following serum induction in HCC2429 stably transfected with DN and VC. Higher levels of MKP-1 were observed in the DN clones ( FIG. 10C ). This finding suggests that Notch3 also modulates MAPK activation through MKP-1 transcriptional control.
- EGFr Inhibitors Enhance the Anti-proliferation Effect of Notch3 Inhibition.
- the inventors previously showed that Notch3 expression positively correlates with EGFR expression in our tumor tissue array.
- the inventors hypothesized that Notch3 acts synergistically with the EGFR pathway in the promotion and the survival of lung cancer cells and that combining inhibitors of both pathways will have synergistic therapeutic value.
- the inventors examined whether inhibiting the Notch3 pathway increases tumor inhibition when lung cancer cells are treated with an EGFR tyrosine kinase inhibitor, AG1478.
- Notch3 activation may decrease a tumor's dependence on the EGF pathway and thus further decrease the sensitivity to EGFR tyrosine kinase inhibitors.
- Synergism can also be observed when the ⁇ -secretase inhibitor L-685,458 is added to AG1478 ( FIG. 11B ).
- the inventors also demonstrate additive effects by combining MRK003, another ⁇ -secretase inhibitor, with an EGFR inhibitor ( FIGS. 12A , 12 B). From a therapeutic standpoint, Notch3 is a good target for therapeutic intervention both alone and in combination with growth factor receptor inhibitors. Since about 80% of lung carcinomas express EGFR, but far fewer respond to kinase inhibition, our data suggest that adding a Notch3 inhibitor will improve the response rate in patients treated with EGFR inhibitors.
- FIG. 13 is a representative experiment showing that the peptides induce apoptosis by Annexin V staining through screening using an FMAT system.
- Each of 155 different peptides were assayed in quadruplicate, and only those peptides that produced a significant increase of fluorescence signal in all 4 wells were considered potentially positive or capable of inducing apoptosis.
- the bar graphs here reflect fluorescence counts.
- FIG. 14A shows HCC2429 treated with Notch3 peptides N16, N17, N102, N103, N132, with induction of apoptosis by peptides compared to control. Treatment with peptides also reduced transcription of Notch3-dependent gene Hey1 as determined by real-time RT-PCR ( FIG. 14B ). Of note, N17 peptide both demonstrates highest apoptotic activity and best reduction in Hey1 transcription.
- FIG. 15 shows HEK cells transfected with Jagged1-HA and treated with Notch3 peptides. The peptides were then immunoprecipitated from cell lysate with streptavidin beads and immunoblotted with anti-HA antibody. This suggests that peptide induces apoptosis via binding to ligand Jagged1 and preventing activation of Notch3 receptor.
- FIG. 16 shows an immunoblot that demonstrates that sera from mice #2, 3, 4, 5, 6 can reduced cleavage of Notch3 ICD (Tx) in Notch3 expressing cell line HCC2429 as compared a control (C).
- Recombinant protein representing EGF-like repeats 21-22 and encompassing sequence CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS was used to immunize AJ and BALB/c mice.
- FIG. 17A shows Fc-fusion protein comprised for N16-17 and N132 sequences inhibits Notch3 activation
- FIG. 17B shows that purified recombinant N16-17-Fc protein induces apoptosis as compared to control and Fc control after 40 hrs treatment.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Abstract
The present invention involves the use of peptides from Notch3, and antibodies that recognize epitopes represented by those peptides, as anti-cancer agents. Methods of combination therapy using standard anti-cancer protocols in conjunction with Notch3 peptides and antibodies also are provided.
Description
- This application claims benefit of priority to U.S. Prov. Appln. Ser. No. 60/972,584, filed Sep. 14, 2007, the entire contents of which are hereby incorporated by reference.
- This invention was made with government support under grant no. 2P50 CA090949 awarded by the National Cancer Institute. The government has certain rights in the invention.
- I. Field of the Invention
- The present invention relates to the fields of oncology and molecular biology. More particular the invention relates to the targeting of the Notch3 receptor.
- II. Related Art
- Lung cancer is the most common cause of cancer-related deaths in the United States. The cure rate for patients with lung cancer remains low—15%—and has not changed significantly during the past 30 years (Jermal et al., 2005). A better understanding of the signaling pathways important in driving and maintaining the malignant state allows the identification of new therapeutic targets and is thus imperative for continued progress in the treatment of these patients. Genes involved in cell fate determination often contribute to tumorigenesis when they are aberrantly expressed. The family of Notch receptors is one such family where there are now strong data linking it to cancer pathogenesis.
- All four members of the Notch receptor family are known to be dysregulated in the majority of human cancers. The inventors were the first to link dysregulation of the Notch3 pathway to human lung cancer (Dang et al., 2000). They demonstrated that Notch3 is highly expressed in 40% of all resected lung cancers and that, in the developing lung, constitutive activation of Notch3 results in inhibition of terminal differentiation. Furthermore, they showed that inhibiting this pathway in human lung tumors results in the loss of the malignant phenotype in vitro and tumor inhibition in xenograft models. This anti-tumor effect is enhanced in the presence of low serum and in combination with an EGFr tyrosine kinase inhibitor. Taken together, these data support an important role for Notch3 and its interaction with the EGF and Ras pathways in lung cancer. However, methods for therapeutic intervention in Notch3 related cancers has not yet been reported.
- Thus, in accordance with the present invention, there is provided an isolated and purified peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues. The peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may further be comprised in a pharmaceutically acceptable diluent, buffer or excipient.
- In another embodiment, there is provided a method of inhibiting Notch3 receptor signaling comprising contacting a cell expressing Notch3 with a peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). The cell may be a cancer cell, such as a lung cancer cell and/or an adenocarcinoma. More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues. The peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may further comprise contacting the cell with two or more peptides comprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may also further comprises contacting the cancer cell with a second agent that inhibits cancer cell growth, differentiation, metastasis or drug resistance.
- In yet another embodiment, there is provided a method of treating a subject having a Notch3-expressing cancer comprising administering to said subject a peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). The subject may a human. The cancer cell may be a lung cancer cell and/or an adenocarcinoma. More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues. The peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may further comprise contacting the cell with two or more peptides comprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may also further comprises contacting the cancer cell with a second agent that inhibits cancer cell growth, differentiation, metastasis or drug resistance.
- Also provided are an isolated and purified antibody that binds to an epitope comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8), as well as methods of using such antibodies to inhibit Notch3 receptor signaling in a cell expressing Notch3.
- In still another embodiment, there is provided a method of treating a subject having a Notch3-expressing cancer comprising administering to said subject an antibody that binds to an epitope comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8).
- Yet another embodiment comprises a pharmaceutical formulation comprising two or more of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10) including three, four, five, six, seven or all of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
- It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
- The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
- These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.
- The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein:
-
FIGS. 1A-B . Diagram demonstrates the major steps of the canonical Notch pathway. (FIG. 1A ) Binding of a DSL ligand triggers proteolysis (S2 and S3) of the Notch receptor and releases the C-terminal NICD fragment. (FIG. 1B ) NICD translocates to the nucleus, recruits coactivators, and binds to CSL factors to promote target gene transcription. In the absence of NICD, the CSL factor associates with the corepressor complex and inhibits target gene transcription. -
FIGS. 2A-B . Proposed mechanisms of antagonistic and cooperative interactions between Ras and Notch pathways. (FIG. 2A ) Notch antagonizes Ras signaling by preventing expression of active EGF (1). Notch activation can also directly inhibit Ras activity (2) and induce expression MAPK inhibitors (3). (FIG. 2B ) Ras activation in turn can induce expression of Notch inhibitors (4). Paradoxically, Ras activation has also been shown to induce DSL ligand expression (5). These observation further support the hypothesis that Notch activation is highly context dependent. However, in a majority of cancers, Notch and ras appear to be cooperative. Adapted from Sundaram (2005). -
FIG. 3 . Immunohistochemistry with an antibody to extracellular domain of Notch3. Cytoplasmic and membranous staining is observed in squamous cell carcinoma (A) and adenocarcinoma (C) of the lung as compared to aneuroendocrine tumor (B) and normal lung (D). In panel B, slight staining is seen in blood vessels (arrow) within the tumor, consistent with other studies demonstrating normal staining of Notch3 in blood vessels -
FIGS. 4A-D . Notch3 alters lung morphology of SP-C-N3IC transgenic mice at E18.5. Five μm-thick lung sections of wild-type littermate controls (FIGS. 4A , 4C) show that epithelial layer of terminal airways are thin and comprised mostly of type I pneumocytes. The terminal lung epithelium of transgenic embryos (FIGS. 4B , 4D) demonstrates severe metaplasia, composed mostly of undifferentiated cuboidal cells (arrowheads). No type I pneumocyte was found. The mesenchyme is abnormally abundant compared to that seen in the wild-type littermate. Bars=50 μm; br., bronchiole; m, mesenchyme; v, vessel. -
FIGS. 5A-B . Notch inhibition inhibits the tumor phenotype. (FIG. 5A ) Inhibition of the Notch3 signaling pathway markedly reduces the size of the colonies formed in soft agar (panel B), compared with vector controls (panel A) in HCC2429 and H460. (FIG. 5B ) In serum-starved conditions, the growth of the DN transfectant is severely inhibited in comparison with that of VC. However, with the addition of exogenous growth factors, the growth rate is equal to that of VC. -
FIG. 6 . Knocking out Notch3 with siRNA reduces focus formation. The pSuper vectors expressing Notch3 siRNA and a mouse Notch3 sequence were transfected into HCC2429, a human lung cancer cell line. The cells were selected with puramycin, then stained with crystal violet after 4 weeks. -
FIGS. 7A-D . A γ-secretase inhibitor inhibits tumor cells and Notch3 processing. (FIG. 7A ) GSI inhibits HCC2429 cells in a serum-dependent manner. HCC2429 cells are sensitive to GSI, and the sensitivity increases in low serum, similarly to that observed with clones expressing the DN construct. Tumor cells treated with DMSO alone had no change in cell viability. (FIG. 7B ) Inhibition of S3 proteolytic processing results in the decrease of Notch3 intracellular domain (N3ICD) and accumulation of S2 product (N3ΔE) after 3 hours. (FIG. 7C ) In this experiment, HCC2429 was stably transfected with plasmid vector expressing Notch3 siRNA. Control C is the parental HCC2429, whereas siRNA-C clones N3 clones FIG. 7D ). Loss of Notch3 results in no loss in cell survival when treated with MRK003. -
FIGS. 8A-D . γ-secretase inhibitor MRK003 demonstrates anti-tumor activity in vivo. (FIG. 8A ) Xenografts injected with HCC2429 were treated with MRK003 once the tumors became palpable. After 2 weeks of treatment, the inventors observed about a 50% reduction in tumor size. (FIG. 8B ) Loss of activated Notch3 (N3ICD) can be seen in tumor treated with MRK003. Histological examination of resected tumors from xenografts at the end of treatment. Marked necrosis can be seen in the MRK003 treated animal (FIG. 8D ) as compare to control (FIG. 8C ). -
FIGS. 9A-C . Inhibition of Notch3 increases apoptosis. (FIG. 9A ) After 72 hours of exogenous growth factor deprivation, cell lines transfected with DN show a higher percentage of apoptosis as measured by Apo-BrdU analysis. (FIG. 9B ) Expression levels of phospho-Akt protein decrease in the Notch3-overexpressing cell line HCC2429 when it is stably transfected with the DN construct, particularly with serum starvation. (FIG. 9C ) Transfection with Notch3 SiRNA resulted in loss of Bcl-xL expression and induction of apoptotic product PARP. -
FIGS. 10A-C . Notch3 crosstalks with the MAPK pathway. (FIG. 10A ) Inhibition of Notch3 signaling in HCC2429 downregulates phospho-p44/42 (ERK?) under serum-starved conditions and after induction with 10% FCS. (FIG. 10B ) When the immortalized lung epithelial cell line BEAS-2B was transfected with the DA construct, the inventors observed higher levels of phospho-p44/p42 under serum-starved conditions as well as after serum induction. (FIG. 10C ) One mechanism of MAPK modulation includes transcriptional regulation of MKP1 in HCC2429. The DN clones demonstrate significantly higher transcriptional level of MKP1 under serum-starved conditions and at 30 minutes and 1 hour after serum induction, when compared with VC (*). -
FIGS. 11A-B . Notch3 modulates the EGF pathway and increases sensitivity to an EGFr inhibitor. (FIG. 11A ) In HCC2429, inhibition of the Notch3 pathway increases sensitivity to AG1478 nearly 40-fold. In H460, a cell line that is markedly resistant to AG1478 (IC50=23.8 μM) when compared to HCC2429 (IC50=8.3 μM), inhibition of Notch3 also increases sensitivity to the inhibitor. (FIG. 11B ) A similar observation is made in H460 when the inventors combine AG1478 with L-685,458, a γ-secretase inhibitor, further supporting the hypothesis that EGF cooperates with the Notch pathway in oncogenesis. -
FIGS. 12A-B . In HCC2429 lung cancer cell line, Notch inhibitor MRK003 enhances the effect of EGFR inhibitor AG1478 on colony formation in soft agar. (FIG. 12A ) Photographs showing that MRK003 not only decreases colony formation, but also enhances the effect of AG1478 on growth. (FIG. 12B ) Graph depicts the quantitative decrease in colony formation. -
FIG. 13 . Notch3 peptides induce apoptosis. Representative experiment showing that the peptides induce apoptosis by Annexin V staining through screening using an FMAT system. Each of 155 different peptides were assayed in quadruplicate, and only those peptides that produced a significant increase of fluorescence signal in all 4 wells were considered potentially positive or capable of inducing apoptosis. The bar graphs here reflect fluorescence counts. Sequences N102: CATAV, N103: CFHGAT, N105: CVSNP, N132: CLNGGS. -
FIG. 14A-B . Notch3 peptides induce apoptosis and inhibit Notch3-regulated gene Hey1. (FIG. 14A ) HCC2429 was treated with Notch3 peptides N16, N17, N102, N103, N132. Induction of apoptosis by peptides is observed as compared to control. MRK003-treated cell is used as positive control. After treatment, cells were labeled with annexin V and detected using flow cytometry. (FIG. 14B ) Treatment with peptides also reduced transcription of Notch3-dependent gene Hey1 as determined by real-time RT-PCR. Of note, N17 peptide both demonstrates highest apoptotic activity and best reduction in Hey1 transcription. MRK003-treated cells were used as positive control. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N102: CATAV, N103: CFHGAT, N132: CLNGGS. -
FIG. 15 . Notch3 Peptides interrupt signaling through binding to ligand Jagged1. HEK cells were transfected with Jagged1-HA and treated with Notch3 peptides. The peptides were then immunoprecipitated from cell lysate with streptavidin beads and immunoblotted with anti-HA antibody. No: no input; C: control peptide. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N102: CATAV, N103: CFHGAT, N132: CLNGGS. -
FIG. 16 . Sera from mice immunized with Notch3 recombindant protein inhibit Notch3 activation. Immunoblot demonstrates sera frommice # -
FIGS. 17A-B . Recombinant Fc-fusion Notch3 proteins inhibit Notch3 activation and induces apoptosis in vitro. (FIG. 17A ) Fc-fusion protein comprised for N16-17 and N132 sequences inhibits Notch3 activation. HCC2429 was treated with purified Fc-fusion protein 10 μg/ml for 24 hrs. (FIG. 17B ) Purified recombinant N16-17-Fc protein induces apoptosis as compared to control and Fc control after 40 hrs treatment. Apoptosis was determined by percentage of annexin V positive cells. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N132: CLNGGS. - Many genes that are important in tumor initiation, progression, or survival play crucial roles in normal development. The Notch receptors are members of an evolutionarily conserved family that is essential for the control of cell fate determination during the development of many multicellular organisms. The core components of the Notch pathway are listed in Table 1. Their functions were first discovered in Drosophila melanogaster almost 80 years ago, when a heterozygous deletion was found to result in “notches” at the wing margins. Mutations in Notch genes alter cell fate determination, causing cells destined to become epidermis to instead give rise to neural tissue (reviewed in Artavanis-Tsakonas, 1999). Notch signaling is classically divided into two fundamental types: inductive and lateral signaling. Induction occurs between two nonequivalent cells, where one cell expresses the receptor and the other expresses the ligand, and through their interaction one cell adopts a different fate. In contrast, lateral signaling occurs between equivalent cells, and through competitive inhibition, adjacent cells are forced to follow a different fate. The role of Notch pathway signaling in mammals has been studied extensively in lymphogenesis, ondotogenesis, neurogenesis, hair and sensory development (Beatus and Leandahl, 1998; Mitsiadis et al., 1998; Lanford et al., 1999; Robey et al., 1996).
-
TABLE 1 Core components of the Notch pathway in worms, flies, and mammals Component C. elegans Drosophila Mammals DSL Ligands LAG-2, APX-1, DSL-1 Delta, Serrate Delta-like-1, -3 and -4 Jagged1, Jagged 2 Notch Receptors LIN-12, Glp-1 Notch Notch 1-4 CSL Factors LAG-1 Suppressor of Hairless CBF-1 Corepressor Hairless, Groucho, SMRT, NcoR, CIR dCTBP, SMRTER Coactivators Mastermind Mastermind Target Genes Enhancer of Split, Hey HES1-7, Hey1-2 *adapted from Sundaram (2005). - Notch Plays A Key Role In Vascular Development and Homeostasis. In adult mammals, the expression of Notch receptors is restricted to the vascular systems. Mice with targeted mutations of the Notch pathway components, such as Jagged1, Delta-like-1 and the Notch1 receptor, die during embryogenesis from defects in vascular morphogenesis (Krebs et al., 2000; Xue et al., 1999; Hrabe de Angelis et al., 1997). While Notch3−/− mice are fertile and viable, these adult mice exhibit structural defects in the distal arteries and arterial myogenic response, reflecting the lack of proper development in vascular smooth muscle cells (Domenga et al., 2004). These observations indicate that the Notch signaling pathway is important in both vascular development and homeostasis. Not surprisingly, many of the processes involved in embryonic vascular development are mirrored in tumor angiogenesis. For example, induction of Notch ligand Jagged1 promotes capillary-like sprout formation in tumor cells (Zeng et al., 2005). Finally, inhibition of Notch activation by γ-secretase inhibitors also inhibits angiogenesis and tumor proliferation (Paris et al., 2005; Williams et al., 2005). These observations support a role of Notch pathway in normal and tumor angiogenesis.
- Activation of Notch Signaling requires Proteolytic Cleavage of the Receptor. While Drosophila possesses a single Notch gene, there are four members of the Notch family in mammals: Notch1 (TAN1), Notch2, Notch3 and Notch4/Int-4. The core components of the Notch pathway are listed in Table 1. Notch is expressed on cell surfaces as a single-pass, heterodimeric receptor. The ligands are also transmembrane proteins of the DSL (Delta/Serrate/LAG-2) family that can be expressed not only on adjacent cells but also on the very same cell expressing the Notch receptors. Receptor-ligand interaction triggers proteolysis at the extracellular S2 site near the transmembrane domain and at the S3 site (
FIG. 1 ). A TNF-α converting enzyme (TACE) and a presenillin-1-dependent γ-secretase are believed to be responsible for the proteolytic processing at sites S2 and S3, respectively. The final cleavage releases the C-terminal, intracellular domain (NICD), which then translocates to the nucleus, recruits coactivators such as mastermind and p300, and binds to CSL (CBF/Suppressor of Hairless/LAG-1) factors. In the absence of Notch signaling, CSL proteins in association with corepressors repress target gene transcription. Thus, Notch signaling causes a switch from transcriptional repression to transcriptional activation of CSL target genes (a review of Notch processing in Mumm and Kopan (2000). - The Notch Signaling Pathway Is Oncogenic. Many key pathways in development play important roles in tumorigenesis when altered. Notch1 was first identified in association with a t(7:9) translocation found in a subset of human T-cell acute lymphoblastoid leukemias (T-ALLs) (Ellisen et al., 1991). While less than 1% of human T-ALLs exhibit the t(7:9), Notch activating mutations have been observed in 50% of human T-ALLs (Weng et al., 2004; Ma et al., 1999). The expression of the constitutively activated intracellular domain (NICD) of Notch1 in bone marrow cells confers an oncogenic phenotype (Pear et al., 1996). Similar observations have been made linking Notch family members with cancer pathogenesis as well (Jhappan et al., 1992; Rohn et al., 1996). Constitutive activation of Notch3 in transgenic mice results in T-cell lymphoblastic leukemia, and in human T-ALL, loss of Notch3 expression correlates with clinical remission Bellavia et al., 2002; Bellavia et al., 2000). Moreover, activated Notch3 confers resistance to apoptosis and loss of contact inhibition in smooth muscle cells (Wang et al., 2002; Sweeney et al., 2004; Campos et al., 2002). Notch3 has been found to be highly expressed in other tumors, including lung, pancreatic and ovarian carcinoma, using gene expression microarray (Dang et al., 2000; Miyamoto et al., 2003; Lu et al., 2004). Similar studies demonstrate correlations between aberrant Notch ligand/receptor expression and tumor development in various systems (Miyamoto et al., 2003; Santagata et al., 2004; Purow et al., 2005; Callahan and Egan, 2004). The inventors published data demonstrating that inhibition of Notch3 activation using a dominant-negative receptor reduces tumor phenotype (Haruki et al., 2005). Taken together, these observations suggest that the Notch pathway is functionally significant in solid tumors and can serve as a target for therapeutic intervention.
- Notch Crosstalks with the Ras Pathway. In both mammals and invertebrates such as Drosophila and C. elegans, Notch receptors signal primarily by the binding to members of the CSL family of transcription factors and related transcription co-activators. However, Notch is known to interact with other pathways including the Wingless/B-catenin and NF-κB pathways (Johnston and Edgar, 1998; Oswald et al., 1998). One pathway that plays prominently in both development and neoplastic transformation is the EGF/ras/MAPK pathway. Notch has been shown in developing organisms to antagonize EGF signaling in cell fate determination through modulation of the MAPK pathway (Faux et al., 2001; Ahmad and Dooley, 1998; Berset et al., 2001). In other cases, however, the Ras and Notch pathways cooperate in promoting certain cell fates (Yoo et al., 2004). As in flies and worms, specific outcomes of EGF and Notch pathways in mammals are context dependent. In mammals, Wang et al. demonstrated that Notch3 induces phosphorylation of ERK1/ERK2 (p44/p42) in vascular smooth muscle cells (Wang et al., 2002). Current evidence indicates that malignant transformation by Notch requires activated Ras (Haruki et al., 2005; Fitzgerald et al., 2000). On the other hand, activated Notch1 was found to inhibit Fgf-dependent malignant transformation of NIH3T3 cells (Small et al., 2003). While further work is required to fully understand the mechanism of interactions between Ras and Notch pathways, the preliminary data demonstrate a cooperative relationship between Notch3 signaling and the ras pathway. This observation suggests that combinatorial therapeutic approach will have better efficacy in the treatment of patients with lung cancers.
FIG. 2 summarizes known potential Notch-ras interactions in both the development and cancer context. - γ-Secretase Inhibitors Demonstrate Antitumor Effects. Proteolytic processing of Notch receptors following ligand binding is necessary for their activation. The final proteolytic cleavage by the γ-secretase protein complex releases the Notch intracellular domain required for target gene transcription. Thus, pharmacologic intervention that inhibits the activity of any of the proteases can potentially inhibit tumor growth in Notch-dependent cancer. Interestingly, at the same time that presenilins were shown to be essential for Notch signaling, they were discovered as susceptibility loci for Alzheimer's disease (Levitan and Greenwald, 1995). The pathogenesis of Alzheimer's disease is believed to be the accumulation of amyloid β-peptides (Aβ) and formation of amyloid plaques. These peptides are derived from the proteolytic processing of the β-amyloid precursor protein (APP) through an intermediate fragment (C99) by γ-secretases (Dovey et al., 2001). Given the great need for better treatment of patients with Alzheimer's disease, inhibitors targeting γ-secretase are being aggressively pursued by many pharmaceutical companies. Predictably, many of these compounds were found to inhibit Notch processing as well. Furthermore, γ-secretase inhibitors block Notch activation and induce apoptosis in multiple cancer cell lines (Qin et al., 2004; Curry et al., 2005; Alves da Costa, 2004). In vivo, these compounds inhibit angiogenesis and tumor growth (Paris et al., 2005). These inhibitors are known to have non-Notch targets, such as erb-4 and CD44, but our data suggest that Notch inhibition may be a component of the observed antitumor effects (Pelletier et al., 2006; Linggi et al., 2006). However, from a practical standpoint, since erb-4 and CD44 are known to be oncogenic, these inhibitors may actually have increased efficacy by virtue of their multiple targets. In fact, a γ-secretase inhibitor by Merck & Co., Inc, is currently in Phase I trials for patients with metastatic or locally advanced breast cancer and for patients with T-cell acute leukemias.
- A. Features of the Polypeptide
- Notch3 is a 2321 amino acid protein (243659 Da Q9UM47; SEQ ID NO:2) that exists as a heterodimer of a C-terminal fragment (TM) and an N-terminal fragment (EC) which are probably linked by disulfide bonds. It has been shown to iteract with MAML1, MAML2 and MAML3 which act as transcriptional coactivators for NOTCH3. It is localized in the cell membrane and is a single-pass type I membrane protein. Following proteolytical processing, the notch intracellular domain (NICD) causes translocation to the nucleus. Its only known post-translational modification is glycosylation.
- Notch3 functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 to regulate cell-fate determination. Upon ligand activation through the released NICD, it forms a transcriptional activator complex with CBF-1 and activates genes of the enhancer of split locus. As discussed above, it has effects the implementation of differentiation, proliferation and apoptotic programs. It is located at 19p13.2-p13.1.
- B. Peptides
- Notch3 peptides will comprise molecules of 5 to no more than about 50 residues in length. A particular length may be less than 39 residues, less than 35 residues, less than 30 residues, less than 25 residues, less than 20 residues, less than 15 residues, or less than 13, including 5, 6, 7, 8, 9, 10, 11 or 12 residues, and ranges of 5-11 residues, 5-15 residues, 5-20 residues, 5-25 residues, 5-30 residues, 5-35 residues, 5-38 residues, or 5-40 residues. The peptides may be generated synthetically or by recombinant techniques, and are purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration), as described in further detail below.
- The peptides may be labeled using various molecules, such as fluorescent, chromogenic or colorimetric agents. The peptides may also be linked to other molecules, including other anti-cancer agents. The links may be direct or through distinct linker molecules. The linker molecules in turn may be subject, in vivo, to cleavage, thereby releasing the agent from the peptide. Peptides may also be rendered multimeric by linking to larger, and possibly inert, carrier molecules.
- C. Variants
- Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
- Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
- The following is a discussion based upon changing of the amino acids of a protein to create an equivalent or improved molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 2 shows the codons that encode particular amino acids.
-
TABLE 2 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU - In making substitutional variants, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
- Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
- It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
- It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine *−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
- It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
- As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al., (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used, in conjunction with the principles outline above, to engineer second generation molecules having many of the natural properties of Notch3, but with altered and even improved characteristics.
- D. Fusions
- A specialized kind of insertional variant is the fusion protein. This molecule generally has all or a substantial portion (e.g., an intracellular, transmembrane or extracellular domain) of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide. For example, fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
- E. Purification of Proteins/Peptides
- It will be desirable to purify Notch3 or fragments thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
- Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term “purified protein or peptide” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
- Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
- Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a “-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
- Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
- There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
- It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
- High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
- Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
- Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
- A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fuctose will bind to lectins from lotus.
- The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
- F. Synthesis
- Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
- G. Antigen Compositions
- The present invention also provides for the use of Notch3 proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either Notch3, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Particular agents are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
- The present invention also provides, in another embodiment, genes encoding Notch3 or fragments (peptides) thereof. A gene for the human Notch3 molecule has been identified. The present invention is not limited in scope to this gene, however, as one of ordinary skill in the could readily identify related homologs in various other species (e.g., mouse, rat, rabbit, dog. monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species).
- In addition, it should be clear that the present invention is not limited to the specific nucleic acids disclosed herein. As discussed below, a “Notch3 gene” may contain a variety of different bases and yet still produce a corresponding polypeptide that is functionally indistinguishable from, and in some cases structurally identical to, the human gene disclosed herein.
- Similarly, any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid. In addition to therapeutic considerations, cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate or enhance the function of Notch3.
- A. Nucleic Acids Encoding Notch3
- Nucleic acids according to the present invention may encode an entire Notch3 coding sequence (Accession No. U97669; SEQ ID NO:1), a domain of Notch3 that expresses a tumor suppressing function, or any other fragment of the Notch3 sequences set forth herein. The nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as “mini-genes.” At a minimum, these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
- The term “cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.
- It also is contemplated that a given Notch3 from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1, above).
- As used in this application, the term “a nucleic acid encoding a Notch3” refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid. In certain embodiments, the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:2. The term “as set forth in SEQ ID NO:2” means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:2. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
- Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO:2. Sequences that are essentially the same as those set forth in SEQ ID NO:2 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:2 under standard conditions.
- The DNA segments of the present invention include those encoding biologically functional equivalent Notch3 proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
- B. Oligonucleotide Probes and Primers
- Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:2. Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules. As used herein, the term “complementary sequences” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:2 under relatively stringent conditions such as those described herein. Such sequences may encode the entire Notch3 protein or functional or non-functional fragments thereof.
- Alternatively, the hybridizing segments may be shorter oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 114 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
- Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
- In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, at temperatures between approximately 20° C. to about 37° C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl2, at temperatures ranging from approximately 40° C. to about 72° C. Formamide and SDS also may be used to alter the hybridization conditions.
- One method of using probes and primers of the present invention is in the search for genes related to Notch3 or, more particularly, homologs of Notch3 from other species. Normally, the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules. By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered.
- Another way of exploiting probes and primers of the present invention is in site-directed, or site-specific mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
- The technique typically employs a bacteriophage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
- In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double-stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
- The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
- C. Vectors for Cloning, Gene Transfer and Expression
- Within certain embodiments, expression vectors are employed to express the Notch3 polypeptide product, which can then be purified for various uses. In other embodiments, the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
- Throughout this application, the term “expression construct” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
- The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al. (1989) and Ausubel et al. (1994), both incorporated herein by reference.
- The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
- (i) Regulatory Elements
- A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
- A promoter may be one naturally-associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally-occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
- Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. One example is the native Notch3 promoter. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.
- Table 3 lists several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof. Table 4 provides examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.
-
TABLE 3 Promoter and/or Enhancer Promoter/Enhancer References Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.; 1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984 T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.; 1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Sherman et al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Ornitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culotta et al., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin Pinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987; Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM) α1-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al., 1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987; Glue et al., 1988 Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989 -
TABLE 4 Inducible Elements Element Inducer References MT II Phorbol Ester (TFA) Palmiter et al., 1982; Haslinger et Heavy metals al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammary Glucocorticoids Huang et al., 1981; Lee et al., tumor virus) 1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI) × Tavernier et al., 1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 Gene A23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb Interferon Blanar et al., 1989 HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a, 1990b Antigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 Tumor Necrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene - The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the
somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996). Tumor specific promoters also will find use in the present invention. Some such promoters are set forth in Tables 4 and 5. -
TABLE 5 Candidate Tissue-Specific Promoters for Cancer Gene Therapy Cancers in which promoter Normal cells in which Tissue-specific promoter is active promoter is active Carcinoembryonic antigen Most colorectal carcinomas; Colonic mucosa; gastric (CEA)* 50% of lung carcinomas; 40-50% mucosa; lung epithelia; of gastric carcinomas; eccrine sweat glands; cells in most pancreatic carcinomas; testes many breast carcinomas Prostate-specific antigen Most prostate carcinomas Prostate epithelium (PSA) Vasoactive intestinal peptide Majority of non-small cell Neurons; lymphocytes; mast (VIP) lung cancers cells; eosinophils Surfactant protein A (SP-A) Many lung adenocarcinomas Type II pneumocytes; Clara cells Human achaete-scute Most small cell lung cancers Neuroendocrine cells in lung homolog (hASH) Mucin-1 (MUC1)** Most adenocarcinomas Glandular epithelial cells in (originating from any tissue) breast and in respiratory, gastrointestinal, and genitourinary tracts Alpha-fetoprotein Most hepatocellular Hepatocytes (under certain carcinomas; possibly many conditions); testis testicular cancers Albumin Most hepatocellular Hepatocytes carcinomas Tyrosinase Most melanomas Melanocytes; astrocytes; Schwann cells; some neurons Tyrosine-binding protein Most melanomas Melanocytes; astrocytes, (TRP) Schwann cells; some neurons Keratin 14 Presumably many squamous Keratinocytes cell carcinomas (e.g., Head and neck cancers) EBV LD-2 Many squamous cell Keratinocytes of upper carcinomas of head and neck digestive Keratinocytes of upper digestive tract Glial fibrillary acidic protein Many astrocytomas Astrocytes (GFAP) Myelin basic protein (MBP) Many gliomas Oligodendrocytes Testis-specific angiotensin- Possibly many testicular Spermatazoa converting enzyme (Testis- cancers specific ACE) Osteocalcin Possibly many osteosarcomas Osteoblasts -
TABLE 6 Candidate Promoters for Tissue-Specific Targeting of Tumors Cancers in which Promoter Normal cells in which Promoter is active Promoter is active E2F-regulated promoter Almost all cancers Proliferating cells HLA-G Many colorectal carcinomas; Lymphocytes; monocytes; many melanomas; possibly spermatocytes; trophoblast many other cancers FasL Most melanomas; many Activated leukocytes: pancreatic carcinomas; most neurons; endothelial cells; astrocytomas possibly many keratinocytes; cells in other cancers immunoprivileged tissues; some cells in lungs, ovaries, liver, and prostate Myc-regulated promoter Most lung carcinomas (both Proliferating cells (only some small cell and non-small cell); cell-types): mammary most colorectal carcinomas epithelial cells (including non- proliferating) MAGE-1 Many melanomas; some non- Testis small cell lung carcinomas; some breast carcinomas VEGF 70% of all cancers Cells at sites of (constitutive overexpression in neovascularization (but unlike many cancers) in tumors, expression is transient, less strong, and never constitutive) bFGF Presumably many different Cells at sites of ischemia (but cancers, since bFGF unlike tumors, expression is expression is induced by transient, less strong, and ischemic conditions never constitutive) COX-2 Most colorectal carcinomas; Cells at sites of inflammation many lung carcinomas; possibly many other cancers IL-10 Most colorectal carcinomas; Leukocytes many lung carcinomas; many squamous cell carcinomas of head and neck; possibly many other cancers GRP78/BiP Presumably many different Cells at sites of ishemia cancers, since GRP7S expression is induced by tumor-specific conditions CarG elements from Egr-1 Induced by ionization Cells exposed to ionizing radiation, so conceivably most radiation; leukocytes tumors upon irradiation - A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
- (ii) IRES
- In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′-methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, herein incorporated by reference).
- (iii) Multi-Purpose Cloning Sites
- Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference. “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
- (iv) Splicing Sites
- Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see Chandler et al., 1997, herein incorporated by reference.)
- (v) Termination Signals
- The vectors or constructs of the present invention will generally comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
- In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3′ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.
- Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
- (vi) Polyadenylation Signals
- In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
- (vii) Origins of Replication
- In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.
- (viii) Selectable and Screenable Markers
- In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.
- Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is calorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.
- (ix) Viral Vectors
- The capacity of certain viral vectors to efficiently infect or enter cells, to integrate into a host cell genome and stably express viral genes, have led to the development and application of a number of different viral vector systems (Robbins et al., 1998). Viral systems are currently being developed for use as vectors for ex vivo and in vivo gene transfer. For example, adenovirus, herpes-simplex virus, retrovirus and adeno-associated virus vectors are being evaluated currently for treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, renal disease and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Pat. No. 5,670,488). The various viral vectors described below, present specific advantages and disadvantages, depending on the particular gene-therapeutic application.
- Adenoviral Vectors. In particular embodiments, an adenoviral expression vector is contemplated for the delivery of expression constructs. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein.
- Adenoviruses comprise linear, double-stranded DNA, with a genome ranging from 30 to 35 kb in size (Reddy et al., 1998; Morrison et al., 1997; Chillon et al., 1999). An adenovirus expression vector according to the present invention comprises a genetically engineered form of the adenovirus. Advantages of adenoviral gene transfer include the ability to infect a wide variety of cell types, including non-dividing cells, a mid-sized genome, ease of manipulation, high infectivity and the ability to be grown to high titers (Wilson, 1996). Further, adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner, without potential genotoxicity associated with other viral vectors. Adenoviruses also are structurally stable (Marienfeld et al., 1999) and no genome rearrangement has been detected after extensive amplification (Parks et al., 1997; Bett et al., 1993).
- Salient features of the adenovirus genome are an early region (E1, E2, E3 and E4 genes), an intermediate region (pIX gene, Iva2 gene), a late region (L1, L2, L3, L4 and L5 genes), a major late promoter (MLP), inverted-terminal-repeats (ITRs) and a ψ sequence (Zheng, et al., 1999; Robbins et al., 1998; Graham and Prevec, 1995). The early genes E1, E2, E3 and E4 are expressed from the virus after infection and encode polypeptides that regulate viral gene expression, cellular gene expression, viral replication, and inhibition of cellular apoptosis. Further on during viral infection, the MLP is activated, resulting in the expression of the late (L) genes, encoding polypeptides required for adenovirus encapsidation. The intermediate region encodes components of the adenoviral capsid. Adenoviral inverted terminal repeats (ITRs; 100-200 bp in length), are cis elements, and function as origins of replication and are necessary for viral DNA replication. The ψ sequence is required for the packaging of the adenoviral genome.
- A common approach for generating an adenoviruses for use as a gene transfer vector is the deletion of the E1 gene (E1−), which is involved in the induction of the E2, E3 and E4 promoters (Graham and Prevec, 1995). Subsequently, a therapeutic gene or genes can be inserted recombinantly in place of the E1 gene, wherein expression of the therapeutic gene(s) is driven by the E1 promoter or a heterologous promoter. The E1−, replication-deficient virus is then proliferated in a “helper” cell line that provides the E1 polypeptides in trans (e.g., the human embryonic kidney cell line 293). Thus, in the present invention it may be convenient to introduce the transforming construct at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. Alternatively, the E3 region, portions of the E4 region or both may be deleted, wherein a heterologous nucleic acid sequence under the control of a promoter operable in eukaryotic cells is inserted into the adenovirus genome for use in gene transfer (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210, each specifically incorporated herein by reference).
- Although adenovirus based vectors offer several unique advantages over other vector systems, they often are limited by vector immunogenicity, size constraints for insertion of recombinant genes and low levels of replication. The preparation of a recombinant adenovirus vector deleted of all open reading frames, comprising a full length dystrophin gene and the terminal repeats required for replication (Haecker et al., 1996) offers some potentially promising advantages to the above mentioned adenoviral shortcomings. The vector was grown to high titer with a helper virus in 293 cells and was capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Helper-dependent viral vectors are discussed below.
- A major concern in using adenoviral vectors is the generation of a replication-competent virus during vector production in a packaging cell line or during gene therapy treatment of an individual. The generation of a replication-competent virus could pose serious threat of an unintended viral infection and pathological consequences for the patient. Armentano et al. (1990), describe the preparation of a replication-defective adenovirus vector, claimed to eliminate the potential for the inadvertent generation of a replication-competent adenovirus (U.S. Pat. No. 5,824,544, specifically incorporated herein by reference). The replication-defective adenovirus method comprises a deleted E1 region and a relocated protein IX gene, wherein the vector expresses a heterologous, mammalian gene.
- Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes and/or subgroups A-F.
Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is becauseadenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector. - As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region. Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210; U.S. Pat. No. 5,824,544). This group of viruses can be obtained in high titers, e.g., 109 to 1011 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. Many experiments, innovations, preclinical studies and clinical trials are currently under investigation for the use of adenoviruses as gene delivery vectors. For example, adenoviral gene delivery-based gene therapies are being developed for liver diseases (Han et al., 1999), psychiatric diseases (Lesch, 1999), neurological diseases (Smith, 1998; Hermens and Verhaagen, 1998), coronary diseases (Feldman et al., 1996), muscular diseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998) and various cancers such as colorectal (Fujiwara and Tanaka, 1998; Dorai et al., 1999), pancreatic, bladder (Irie et al., 1999), head and neck (Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra et al., 1999) and ovarian (Vanderkwaak et al., 1999).
- Retroviral Vectors. In certain embodiments of the invention, the use of retroviruses for gene delivery are contemplated. Retroviruses are RNA viruses comprising an RNA genome. When a host cell is infected by a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated into the chromosomal DNA of infected cells. This integrated DNA intermediate is referred to as a provirus. A particular advantage of retroviruses is that they can stably infect dividing cells with a gene of interest (e.g., a therapeutic gene) by integrating into the host DNA, without expressing immunogenic viral proteins. Theoretically, the integrated retroviral vector will be maintained for the life of the infected host cell, expressing the gene of interest.
- The retroviral genome and the proviral DNA have three genes: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase) and the env gene encodes viral envelope glycoproteins. The 5′ and 3′ LTRs serve to promote transcription and polyadenylation of the virion RNAs. The LTR contains all other cis-acting sequences necessary for viral replication.
- A recombinant retrovirus of the present invention may be genetically modified in such a way that some of the structural, infectious genes of the native virus have been removed and replaced instead with a nucleic acid sequence to be delivered to a target cell (U.S. Pat. No. 5,858,744; U.S. Pat. No. 5,739,018, each incorporated herein by reference). After infection of a cell by the virus, the virus injects its nucleic acid into the cell and the retrovirus genetic material can integrate into the host cell genome. The transferred retrovirus genetic material is then transcribed and translated into proteins within the host cell. As with other viral vector systems, the generation of a replication-competent retrovirus during vector production or during therapy is a major concern. Retroviral vectors suitable for use in the present invention are generally defective retroviral vectors that are capable of infecting the target cell, reverse transcribing their RNA genomes, and integrating the reverse transcribed DNA into the target cell genome, but are incapable of replicating within the target cell to produce infectious retroviral particles (e.g., the retroviral genome transferred into the target cell is defective in gag, the gene encoding virion structural proteins, and/or in pol, the gene encoding reverse transcriptase). Thus, transcription of the provirus and assembly into infectious virus occurs in the presence of an appropriate helper virus or in a cell line containing appropriate sequences enabling encapsidation without coincident production of a contaminating helper virus.
- The growth and maintenance of retroviruses is known in the art (U.S. Pat. No. 5,955,331; U.S. Pat. No. 5,888,502, each specifically incorporated herein by reference). Nolan et al. describe the production of stable high titre, helper-free retrovirus comprising a heterologous gene (U.S. Pat. No. 5,830,725, specifically incorporated herein by reference). Methods for constructing packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphoteric or ecotrophic host ranges, as well as methods of using the recombinant retroviruses to introduce a gene of interest into eukaryotic cells in vivo and in vitro are contemplated in the present invention (U.S. Pat. No. 5,955,331).
- Currently, the majority of all clinical trials for vector-mediated gene delivery use murine leukemia virus (MLV)-based retroviral vector gene delivery (Robbins et al., 1998; Miller et al., 1993). Disadvantages of retroviral gene delivery includes a requirement for ongoing cell division for stable infection and a coding capacity that prevents the delivery of large genes. However, recent development of vectors such as lentivirus (e.g., HIV), simian immunodeficiency virus (SIV) and equine infectious-anemia virus (EIAV), which can infect certain non-dividing cells, potentially allow the in vivo use of retroviral vectors for gene therapy applications (Amado and Chen, 1999; Klimatcheva et al., 1999; White et al., 1999; Case et al., 1999). For example, HIV-based vectors have been used to infect non-dividing cells such as neurons (Miyatake et al., 1999), islets (Leibowitz et al., 1999) and muscle cells (Johnston et al., 1999). The therapeutic delivery of genes via retroviruses are currently being assessed for the treatment of various disorders such as inflammatory disease (Moldawer et al., 1999), AIDS (Amado and Chen, 1999; Engel and Kohn, 1999), cancer (Clay et al., 1999), cerebrovascular disease (Weihl et al., 1999) and hemophilia (Kay, 1998).
- Herpesviral Vectors. Herpes simplex virus (HSV) type I and type II contain a double-stranded, linear DNA genome of approximately 150 kb, encoding 70-80 genes. Wild type HSV are able to infect cells lytically and to establish latency in certain cell types (e.g., neurons). Similar to adenovirus, HSV also can infect a variety of cell types including muscle (Yeung et al., 1999), ear (Derby et al., 1999), eye (Kaufman et al., 1999), tumors (Yoon et al., 1999; Howard et al., 1999), lung (Kohut et al., 1998), neuronal (Gamido et al., 1999; Lachmann and Efstathiou, 1999), liver (Miyatake et al., 1999; Kooby et al., 1999) and pancreatic islets (Rabinovitch et al., 1999).
- HSV viral genes are transcribed by cellular RNA polymerase II and are temporally regulated, resulting in the transcription and subsequent synthesis of gene products in roughly three discernable phases or kinetic classes. These phases of genes are referred to as the Immediate Early (IE) or alpha genes, Early (E) or beta genes and Late (L) or gamma genes. Immediately following the arrival of the genome of a virus in the nucleus of a newly infected cell, the IE genes are transcribed. The efficient expression of these genes does not require prior viral protein synthesis. The products of IE genes are required to activate transcription and regulate the remainder of the viral genome.
- For use in therapeutic gene delivery, HSV must be rendered replication-defective. Protocols for generating replication-defective HSV helper virus-free cell lines have been described (U.S. Pat. No. 5,879,934; U.S. Pat. No. 5,851,826, each specifically incorporated herein by reference in its entirety). One IE protein, Infected Cell Polypeptide 4 (ICP4), also known as
alpha 4 or Vmw175, is absolutely required for both virus infectivity and the transition from IE to later transcription. Thus, due to its complex, multifunctional nature and central role in the regulation of HSV gene expression, ICP4 has typically been the target of HSV genetic studies. - Phenotypic studies of HSV viruses deleted of ICP4 indicate that such viruses will be potentially useful for gene transfer purposes (Krisky et al., 1998a). One property of viruses deleted for ICP4 that makes them desirable for gene transfer is that they only express the five other IE genes: ICP0, ICP6, ICP27, ICP22 and ICP47 (DeLuca et al., 1985), without the expression of viral genes encoding proteins that direct viral DNA synthesis, as well as the structural proteins of the virus. This property is desirable for minimizing possible deleterious effects on host cell metabolism or an immune response following gene transfer. Further deletion of IE genes ICP22 and ICP27, in addition to ICP4, substantially improve reduction of HSV cytotoxicity and prevented early and late viral gene expression (Krisky et al., 1998b).
- The therapeutic potential of HSV in gene transfer has been demonstrated in various in vitro model systems and in vivo for diseases such as Parkinson's (Yamada et al., 1999), retinoblastoma (Hayashi et al., 1999), intracerebral and intradermal tumors (Moriuchi et al., 1998), B-cell malignancies (Suzuki et al., 1998), ovarian cancer (Wang et al., 1998) and Duchenne muscular dystrophy (Huard et al., 1997).
- Adeno-Associated Viral Vectors. Adeno-associated virus (AAV), a member of the parvovirus family, is a human virus that is increasingly being used for gene delivery therapeutics. AAV has several advantageous features not found in other viral systems. First, AAV can infect a wide range of host cells, including non-dividing cells. Second, AAV can infect cells from different species. Third, AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. For example, it is estimated that 80-85% of the human population has been exposed to AAV. Finally, AAV is stable at a wide range of physical and chemical conditions which lends itself to production, storage and transportation requirements.
- The AAV genome is a linear, single-stranded DNA molecule containing 4681 nucleotides. The AAV genome generally comprises an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs) of approximately 145 bp in length. The ITRs have multiple functions, including origins of DNA replication, and as packaging signals for the viral genome. The internal non-repeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for viral proteins that allow the virus to replicate and package the viral genome into a virion. A family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and
Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3. - AAV is a helper-dependent virus requiring co-infection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia) in order to form AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus “rescues” the integrated genome, allowing it to replicate and package its genome into infectious AAV virions. Although AAV can infect cells from different species, the helper virus must be of the same species as the host cell (e.g., human AAV will replicate in canine cells co-infected with a canine adenovirus).
- AAV has been engineered to deliver genes of interest by deleting the internal non-repeating portion of the AAV genome and inserting a heterologous gene between the ITRs. The heterologous gene may be functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in target cells. To produce infectious recombinant AAV (rAAV) containing a heterologous gene, a suitable producer cell line is transfected with a rAAV vector containing a heterologous gene. The producer cell is concurrently transfected with a second plasmid harboring the AAV rep and cap genes under the control of their respective endogenous promoters or heterologous promoters. Finally, the producer cell is infected with a helper virus.
- Once these factors come together, the heterologous gene is replicated and packaged as though it were a wild-type AAV genome. When target cells are infected with the resulting rAAV virions, the heterologous gene enters and is expressed in the target cells. Because the target cells lack the rep and cap genes and the adenovirus helper genes, the rAAV cannot further replicate, package or form wild-type AAV.
- The use of helper virus, however, presents a number of problems. First, the use of adenovirus in a rAAV production system causes the host cells to produce both rAAV and infectious adenovirus. The contaminating infectious adenovirus can be inactivated by heat treatment (56° C. for 1 hour). Heat treatment, however, results in approximately a 50% drop in the titer of functional rAAV virions. Second, varying amounts of adenovirus proteins are present in these preparations. For example, approximately 50% or greater of the total protein obtained in such rAAV virion preparations is free adenovirus fiber protein. If not completely removed, these adenovirus proteins have the potential of eliciting an immune response from the patient. Third, AAV vector production methods which employ a helper virus require the use and manipulation of large amounts of high titer infectious helper virus, which presents a number of health and safety concerns, particularly in regard to the use of a herpesvirus. Fourth, concomitant production of helper virus particles in rAAV virion producing cells diverts large amounts of host cellular resources away from rAAV virion production, potentially resulting in lower rAAV virion yields.
- Lentiviral Vectors. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
- Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. The lentiviral genome and the proviral DNA have the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), a protease and an integrase; and the env gene encodes viral envelope glycoproteins. The 5′ and 3′ LTR's serve to promote transcription and polyadenylation of the virion RNA's. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx.
- Adjacent to the 5′ LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.
- Lentiviral vectors are known in the art, see Naldini et al., (1996); Zufferey et al., (1997); U.S. Pat. Nos. 6,013,516; and 5,994,136. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
- Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene, such as the STAT-1α gene in this invention, into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.
- One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
- The vector providing the viral env nucleic acid sequence is associated operably with regulatory sequences, e.g., a promoter or enhancer. The regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences.
- The heterologous or foreign nucleic acid sequence, such as the STAT-1α encoding polynucleotide sequence herein, is linked operably to a regulatory nucleic acid sequence. Preferably, the heterologous sequence is linked to a promoter, resulting in a chimeric gene. The heterologous nucleic acid sequence may also be under control of either the viral LTR promoter-enhancer signals or of an internal promoter, and retained signals within the retroviral LTR can still bring about efficient expression of the transgene. Marker genes may be utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of a marker gene ensures the selection and growth of only those host cells which express the inserts. Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate, etc., and cell surface markers.
- The vectors are introduced via transfection or infection into the packaging cell line. The packaging cell line produces viral particles that contain the vector genome. Methods for transfection or infection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art. Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be linked physically to the packaging genes in the construct.
- Lentiviral transfer vectors Naldini et al. (1996), have been used to infect human cells growth-arrested in vitro and to transduce neurons after direct injection into the brain of adult rats. The vector was efficient at transferring marker genes in vivo into the neurons and long term expression in the absence of detectable pathology was achieved. Animals analyzed ten months after a single injection of the vector showed no decrease in the average level of transgene expression and no sign of tissue pathology or immune reaction (Blomer et al., 1997). Thus, in the present invention, one may graft or transplant cells infected with the recombinant lentivirus ex vivo, or infect cells in vivo.
- Other Viral Vectors. The development and utility of viral vectors for gene delivery is constantly improving and evolving. Other viral vectors such as poxvirus; e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alpha virus; e.g., sindbis virus, Semliki forest virus (Lundstrom, 1999), reovirus (Coffey et al., 1998) and influenza A virus (Neumann et al., 1999) are contemplated for use in the present invention and may be selected according to the requisite properties of the target system.
- In certain embodiments, vaccinia viral vectors are contemplated for use in the present invention. Vaccinia virus is a particularly useful eukaryotic viral vector system for expressing heterologous genes. For example, when recombinant vaccinia virus is properly engineered, the proteins are synthesized, processed and transported to the plasma membrane. Vaccinia viruses as gene delivery vectors have recently been demonstrated to transfer genes to human tumor cells, e.g., EMAP-II (Gnant et al., 1999), inner ear (Derby et al., 1999), glioma cells, e.g., p53 (Timiryasova et al., 1999) and various mammalian cells, e.g., P-450 (U.S. Pat. No. 5,506,138). The preparation, growth and manipulation of vaccinia viruses are described in U.S. Pat. No. 5,849,304 and U.S. Pat. No. 5,506,138 (each specifically incorporated herein by reference).
- In other embodiments, sindbis viral vectors are contemplated for use in gene delivery. Sindbis virus is a species of the alphavirus genus (Garoff and Li, 1998) which includes such important pathogens as Venezuelan, Western and Eastern equine encephalitis viruses (Sawai et al., 1999; Mastrangelo et al., 1999). In vitro, sindbis virus infects a variety of avian, mammalian, reptilian, and amphibian cells. The genome of sindbis virus consists of a single molecule of single-stranded RNA, 11,703 nucleotides in length. The genomic RNA is infectious, is capped at the 5′ terminus and polyadenylated at the 3′ terminus, and serves as mRNA. Translation of a vaccinia virus 26S mRNA produces a polyprotein that is cleaved co- and post-translationally by a combination of viral and presumably host-encoded proteases to give the three virus structural proteins, a capsid protein (C) and the two envelope glycoproteins (E1 and PE2, precursors of the virion E2).
- Three features of sindbis virus suggest that it would be a useful vector for the expression of heterologous genes. First, its wide host range, both in nature and in the laboratory. Second, gene expression occurs in the cytoplasm of the host cell and is rapid and efficient. Third, temperature-sensitive mutations in RNA synthesis are available that may be used to modulate the expression of heterologous coding sequences by simply shifting cultures to the non-permissive temperature at various time after infection. The growth and maintenance of sindbis virus is known in the art (U.S. Pat. No. 5,217,879, specifically incorporated herein by reference).
- Chimeric Viral Vectors. Chimeric or hybrid viral vectors are being developed for use in therapeutic gene delivery and are contemplated for use in the present invention. Chimeric poxyiral/retroviral vectors (Holzer et al., 1999), adenoviral/retroviral vectors (Feng et al., 1997; Bilbao et al., 1999; Caplen et al., 1999) and adenoviral/adeno-associated viral vectors (Fisher et al., 1996; U.S. Pat. No. 5,871,982) have been described.
- These “chimeric” viral gene transfer systems can exploit the favorable features of two or more parent viral species. For example, Wilson et al., provide a chimeric vector construct which comprises a portion of an adenovirus,
AAV 5′ and 3′ ITR sequences and a selected transgene, described below (U.S. Pat. No. 5,871,983, specifically incorporate herein by reference). - The adenovirus/AAV chimeric virus uses adenovirus nucleic acid sequences as a shuttle to deliver a recombinant AAV/transgene genome to a target cell. The adenovirus nucleic acid sequences employed in the hybrid vector can range from a minimum sequence amount, which requires the use of a helper virus to produce the hybrid virus particle, to only selected deletions of adenovirus genes, which deleted gene products can be supplied in the hybrid viral production process by a selected packaging cell. At a minimum, the adenovirus nucleic acid sequences employed in the pAdA shuttle vector are adenovirus genomic sequences from which all viral genes are deleted and which contain only those adenovirus sequences required for packaging adenoviral genomic DNA into a preformed capsid head. More specifically, the adenovirus sequences employed are the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences of an adenovirus (which function as origins of replication) and the native 5′ packaging/enhancer domain, that contains sequences necessary for packaging linear Ad genomes and enhancer elements for the E1 promoter. The adenovirus sequences may be modified to contain desired deletions, substitutions, or mutations, provided that the desired function is not eliminated.
- The AAV sequences useful in the above chimeric vector are the viral sequences from which the rep and cap polypeptide encoding sequences are deleted. More specifically, the AAV sequences employed are the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences. These chimeras are characterized by high titer transgene delivery to a host cell and the ability to stably integrate the transgene into the host cell chromosome (U.S. Pat. No. 5,871,983, specifically incorporate herein by reference). In the hybrid vector construct, the AAV sequences are flanked by the selected adenovirus sequences discussed above. The 5′ and 3′ AAV ITR sequences themselves flank a selected transgene sequence and associated regulatory elements, described below. Thus, the sequence formed by the transgene and flanking 5′ and 3′ AAV sequences may be inserted at any deletion site in the adenovirus sequences of the vector. For example, the AAV sequences are desirably inserted at the site of the deleted E1a/E1b genes of the adenovirus. Alternatively, the AAV sequences may be inserted at an E3 deletion, E2a deletion, and so on. If only the
adenovirus 5′ ITR/packaging sequences and 3′ ITR sequences are used in the hybrid virus, the AAV sequences are inserted between them. - The transgene sequence of the vector and recombinant virus can be a gene, a nucleic acid sequence or reverse transcript thereof, heterologous to the adenovirus sequence, which encodes a protein, polypeptide or peptide fragment of interest. The transgene is operatively linked to regulatory components in a manner which permits transgene transcription. The composition of the transgene sequence will depend upon the use to which the resulting hybrid vector will be put. For example, one type of transgene sequence includes a therapeutic gene which expresses a desired gene product in a host cell. These therapeutic genes or nucleic acid sequences typically encode products for administration and expression in a patient in vivo or ex vivo to replace or correct an inherited or non-inherited genetic defect or treat an epigenetic disorder or disease.
- (x) Non-Viral Transformation
- Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.
- Injection. In certain embodiments, a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, either subcutaneously, intradermally, intramuscularly, intervenously or intraperitoneally. Methods of injection of vaccines are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution). Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985).
- Electroporation. In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384,253, incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.
- Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.
- To effect transformation by electroporation in cells such as, for example, plant cells, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Examples of some species which have been transformed by electroporation of intact cells include maize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987) and tobacco (Lee et al., 1989).
- One also may employ protoplasts for electroporation transformation of plant cells (Bates, 1994; Lazzeri, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application No. WO 9217598, incorporated herein by reference. Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato (Tsukada, 1989).
- Calcium Phosphate. In other embodiments of the present invention, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with
adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990). - DEAE-Dextran: In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
- Sonication Loading. Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonic loading. LTK− fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987).
- Liposome-Mediated Transfection. In a further embodiment of the invention, a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).
- Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).
- In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.
- Receptor-Mediated Transfection. Still further, a nucleic acid may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.
- Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated herein by reference). In certain aspects of the present invention, a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.
- In other embodiments, a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.
- In still further embodiments, the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner.
- F. Expression Systems
- Numerous prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
- The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name M
AX BAC ® 2.0 from INVITROGEN ® and BAC PACK ™ BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®. - Other examples of expression systems include S
TRATAGENE ®'S COMPLETE CONTROL ™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN ®, which carries the T-REX ™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide. - Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented.
- One embodiment of the foregoing involves the use of gene transfer to immortalize cells for the production of proteins. The gene for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions. The gene for virtually any polypeptide may be employed in this manner. The generation of recombinant expression vectors, and the elements included therein, are discussed above. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell in question.
- Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
- A number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
- G. Host Cells
- As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.
- Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5α, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
- Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
- Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
- H. Cell Propagation
- Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth). Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
- Large scale suspension culture of mammalian cells in stirred tanks is a common method for production of recombinant proteins. Two suspension culture reactor designs are in wide use—the stirred reactor and the airlift reactor. The stirred design has successfully been used on an 8000 liter capacity for the production of interferon. Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.
- The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
- The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Non-ionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations. Antibodies are and their uses are discussed further, below.
- In another aspect, the present invention contemplates an antibody that is immunoreactive with a Notch3 molecule of the present invention, or any portion thereof. In particular, the invention contemplates using peptides having the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, optionally linked together or linked to a carrier molecule such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988).
- Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide, of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
- Antibodies, both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
- It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to Notch3-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular Notch3 of different species may be utilized in other useful applications
- In general, both polyclonal and monoclonal antibodies against Notch3 may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other Notch3. They may also be used in inhibition studies to analyze the effects of Notch3 related peptides in cells or animals. Anti-Notch3 antibodies will also be useful in immunolocalization studies to analyze the distribution of Notch3 during various cellular events, for example, to determine the cellular or tissue-specific distribution of Notch3 polypeptides under different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant Notch3, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
- Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988; incorporated herein by reference). More specific examples of monoclonal antibody preparation are give in the examples below.
- As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
- As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
- The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
- MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified Notch3 protein, polypeptide or peptide or cell expressing high levels of Notch3. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
- Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×107 to 2×108 lymphocytes.
- The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
- Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.
- Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).
- Fusion procedures usually produce viable hybrids at low frequencies, around 1×10−6 to 1×10−8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
- The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
- This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
- The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
- The present invention also involves, in another embodiment, the treatment of cancer. The types of cancer that may be treated, according to the present invention, is limited only by the involvement of Notch3. By involvement, it is not even a requirement that Notch3 be mutated or abnormal—the overexpression of this tumor suppressor may actually overcome other lesions within the cell. Thus, it is contemplated that a wide variety of tumors may be treated using Notch3 therapy, including cancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue.
- In many contexts, it is not necessary that the tumor cell be killed or induced to undergo normal cell death or “apoptosis.” Rather, to accomplish a meaningful treatment, all that is required is that the tumor growth be slowed to some degree. It may be that the tumor growth is completely blocked, however, or that some tumor regression is achieved. Clinical terminology such as “remission” and “reduction of tumor” burden also are contemplated given their normal usage.
- A. Peptide Therapy
- Another therapy approach is the provision, to a subject, of Notch3 polypeptide, fragments, synthetic peptides, mimetics or other analogs thereof. The protein/peptide may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer. Formulations would be selected based on the route of administration and purpose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
- B. Antibody Therapy
- Applicants also contemplate the use of antibodies to Notch3, in particular, to epitopes comprised in or represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. Antibodies will be administered according to standard protocols for passive immunotherapy. Administration protocols would generally involve intratumoral, local or regional (to the tumor) administration, as well as systemic administration.
- In addition, the antibody reagent may be altered, such that it will have one or more improved properties. The antibody may be recombinant, i.e., an antibody gene cloned into an expression cassette which is then introduced into a cell in which the antibody gene was not initially created. The antibody may be single chain, a fragment (Fab, Fv, Vh, ScFv), chimeric or humanized.
- C. Combined Therapies with Immunotherapy, Traditional Chemo- or Radiotherapy
- Tumor cell resistance to DNA damaging agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy. One way is by combining such traditional therapies with gene therapy. For example, the herpes simplex-thymidine kinase (HS-tk) gene, when delivered to brain tumors by a retroviral vector system, successfully induced susceptibility to the antiviral agent ganciclovir (Culver et al., 1992). In the context of the present invention, it is contemplated that Notch3 peptide or antibody therapy could be used similarly in conjunction with chemo- or radiotherapeutic intervention. It also may prove effective to combine a Notch3-directed therapy with another cancer therapy.
- To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one would generally contact a “target” cell with a Notch3 peptide or antibody and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with a Notch3 peptide or antibody and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
- Alternatively, the Notch3 peptide or antibody therapy treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and a Notch3 peptide or antibody would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
- It also is conceivable that more than one administration of either Notch3 peptide or antibody or the other agent will be desired. Various combinations may be employed, where Notch3 (peptide or antibody) is “A” and the other agent is “B”, as exemplified below:
-
- A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
- A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A
- A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
Other combinations are contemplated. Again, to achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell.
- Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as “chemotherapeutic agents,” function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. The invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide. In certain embodiments, the use of cisplatin in combination with a Notch3 expression construct is particularly preferred as this compound.
- In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, γ-rays or even microwaves. Alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin. The agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a Notch3 expression construct, as described above.
- Agents that directly cross-link nucleic acids, specifically DNA, are envisaged to facilitate DNA damage leading to a synergistic, antineoplastic combination with Notch3. Agents such as cisplatin, and other DNA alkylating agents may be used. Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m2 for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
- Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for adriamycin, to 35-50 mg/m2 for etoposide intravenously or double the intravenous dose orally.
- Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage. As such a number of nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU), are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.
- Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
- The skilled artisan is directed to “Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
- The inventors propose that the local or regional delivery of Notch3 expression constructs to patients with cancer will be a very efficient method for treating the clinical disease. Similarly, the chemo- or radiotherapy may be directed to a particular, affected region of the subjects body. Alternatively, systemic delivery of expression construct and/or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
- In addition to combining Notch3 therapies with chemo- and radiotherapies, it also is contemplated that combination with other gene therapies will be advantageous. For example, targeting of Notch3 and p53 mutations at the same time may produce an improved anti-cancer treatment. Any other tumor-related gene conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.
- It also should be pointed out that any of the foregoing therapies may prove useful by themselves in treating a Notch3. In this regard, reference to chemotherapeutics and non-Notch3 gene therapy in combination should also be read as a contemplation that these approaches may be employed separately.
- D. Formulations and Routes for Administration to Patients
- Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions—a Notch3 peptide, or antibody—in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
- One will generally desire to employ appropriate salts and buffers to render delivery compositions stable and allow for uptake by target cells. Aqueous compositions of the present invention comprise an effective amount of the Notch3 peptide or antibody to cells/a subject, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
- The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. Of particular interest is direct intratumoral administration, perfusion of a tumor, or administration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed.
- The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- For oral administration the Notch3 peptides or antibodies of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
- The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
- Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
- The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
- Notch3 Is Expressed in Resected Human Lung Cancers. The clinical relevance of a pathway to tumorigenesis often depends on the prevalence of its dysregulation. To begin to assess the prevalence of Notch3 involvement in lung cancer, the inventors investigated the frequency of Notch3 overexpression in resected lung cancer. They used immunohistochemistry (IHC), with a characterized and validated Notch3 antibody recognizing the extracellular domain (Joutel et al., 2000), in resected lung tumor tissues. For this experiment, the inventors used tumor tissue arrays produced as part of the Vanderbilt SPORE initiative. When the Notch3 antibody targeting the extracellular domain was used, a pattern of cytoplasmic and membranous staining was observed in the representative adenocarcinoma and squamous cell carcincoma (
FIG. 3 , panel A and C). No staining was observed in a neuroendocrine tumor and normal lung tissue (panel B and D), suggesting that Notch3 dysregulation is specific to cancer. The staining was scored on a composite scale of 0 to 4 by two independent investigators, including one pathologist. No staining was scored 0, whereas slight positivity equivalent to background stain was scored as 1. Tumors that scored 2 or higher were considered positive. Fractional positivity was nearly 100% in many tumors with high Notch3 expression. Of the 207 resected tumors, 39% had high Notch3 expression (Table 7). Each tumor was represented in triplicate, and the final score per tumor was calculated by averaging the score of three samples. -
TABLE 7 Expression of Notch3 and EGFr By IHC Using Microtissue Arrays Notch3+ EGFr+ N n % n % Adenocarcinomas 87 32 37% 69 79 % Neuroendocrine Carcinoid 10 2 20% 5 50% Large Cell 7 1 14% 2 29% Small Cell 4 1 25% 3 75% Squamous Cell Carcinomas 88 40 45% 81 92% Large Cell 11 4 36% 8 73% 207 80 39% 168 81% - The frequency of Notch3 overexpression in lung tumors is higher than the frequency of HER2/neu (16%) and k-Ras mutations (16%), and comparable to EGFr (13-80%) (Hirsch et al., 2002; Rodenhuis et al., 1997; Meert et al., 2002). No information is available with regard to the frequency of overexpression of other members of the Notch family or their receptors in human lung carcinomas. The lack of high quality antibodies to the Notch receptors, Jagged1, and Delta-like-1, -3 and -4 makes it difficult to determine their frequency in fresh tumor tissues. In both C. elegans and Drosophila, the Notch pathway crosstalks with the EGF pathway in cell fate determination during development. The inventors thus examined the correlation between Notch3 and EGFr expression. The frequency of EGFr expression in our tumor tissue arrays was 81%. 19% of EGFr positive tumors are Notch3 negative, whereas 43% of EGFr positive tumors are also Notch3 positive (p<0.0001 using Pearson correlation coefficient) (Haruki et al., 2005). This highly statistical association suggests that the Notch and EGF pathways are cooperative in lung tumorigenesis.
- Ectopic Notch3 Expression Inhibits Terminal Differentiation in Developing Lungs of Transgenic Mice. To evaluate the potential transforming activity of Notch3 in vivo, the inventors studied the effect of activated Notch3 using a lung-specific, human SP-C promoter (Glasser et al., 1991; Lardelli et al., 1996). The constitutively-activate Notch3 allowed the inventors to assess the effect of ectopic expression of Notch3 without depending on ligand expression in the epithelium, since Jagged1 expression becomes restricted to endothelium as the lung matures (Taichman et al., 2002). Furthermore, constitutive expression of Notch3 also better mimics the many dysregulated pathways observed in cancer. The inventors observed perinatal lethality, and thus no surviving animals expresses the N3IC transgene.
- Because of the crucial role of the Notch family in development, expression of a constitutively active Notch3 transgene could potentially disturb normal lung development. Therefore, the transgenic mice were sacrificed prior to birth, at E18.5. Of the 182 embryos at E18.5 gestation collected from 32 pregnant mothers, 10 were transgenic, as determined by PCR and Southern blot analysis. The inventors observed altered lung morphogenesis, altered terminal sac morphology, and abnormally abundant mesenchyme, and no type I pneumocytes when compared with control mice (
FIGS. 4A-D ). Despite the important role of the Notch signaling pathway in vascular development, no alteration in vasculogenesis was observed in the transgenics using the PECAM-1 antibody (data not shown), suggesting that the mesenchymal differentiation was influenced by the abnormally developing epithelium, and not by alterations of angiogenesis. While the majority of cuboidal cells lining the peripheral airways are pneumocytes based on their TTF1 positivity, they failed to demonstrate markers of mature type II pneumocytes, such as surfactant proteins C and B, markers for Clara cells (Clara cells secretory protein), or neuroendocrine cells (calcitonin gene-related peptide) suggesting that they were immature type II cells (data not shown). - Thus, this transgenic model provides evidence that dysregulation of Notch3 signaling in the developing lung affects morphogenesis and terminal differentiation of the lung epithelium. Prenatal activation of Notch3 in the peripheral epithelium of the lung in our SP-C-N3IC mouse model leads to nonviable newborn pups, making it impossible to evaluate tumor progression. An inducible transgenic model that allows activation of Notch3 signaling in the postnatal period will be more effectively recapitulate the somatic activation of potential oncogenes observed in human lung cancer. This approach is part of the proposal in our R01 funding. Regardless, the ectopic expression of Notch3 appears to inhibit terminal differentiation of type I pneumocytes and results in metaplasia of the immature respiratory epithelium, supporting a potential role of Notch3 in lung cancer formation.
- Notch3 Inhibition Inhibits the Tumor Phenotype. To test their hypothesis that Notch3 plays an important role in the pathogenesis of lung cancer, the inventors used the approach of uncoupling its ligand binding and signaling functions (Rebay et al., 1993). To inhibit Notch3 activation, the inventors created a dominant-negative (DN) construct. One characteristic feature of malignant transformation is the ability of tumor cells to proliferate in the absence of adhesion, as measured by the soft-agar assay for colony formation. The inventors demonstrated that inhibition of Notch3 signaling by the DN construct dramatically decreases the ability of HCC2429 and H460 to form colonies in soft agar (
FIG. 5A ). Furthermore, colonies formed by the DN clones are markedly smaller than those seen with the vector control (VC). Transformed cells often have a relaxed serum or growth factor requirement for proliferation in monolayer culture (Holley, 1975). In serum-free medium, the DN clones failed to proliferate when compared to the vector controls (FIG. 5B ). In summary, these observations demonstrated that inhibiting the Notch pathway using DN constructs reduces the tumor phenotype, supporting an oncogenic role for Notch3 in lung cancer. - Since Jagged1 is known to bind to other Notch receptors, it is possible that the antitumor effect observed when DN receptor is not Notch3-specific, since its mechanism of action is to sequester ligand (Shimizu et al., 2000). To determine whether specific inhibition of Notch3 activation can inhibit tumor phenotype, the inventors used siRNA to specifically knock down Notch3 expression. The inventors show that inhibiting Notch3 results in the lack of focus formation, further supporting the role of Notch3 in lung cancer pathogenesis (
FIG. 6 ). - A γ-Secretase Inhibitor Reduces Proliferation in Lung Cancer Cells. Proteolytic processing of Notch receptors is required for activation. As previously described, three proteolytic cleavage sites are involved in enabling Notch signaling. In the final step, the membrane-associated Notch fragment is cleaved within its transmembrane domain by a γ-secretase-containing protein complex. Mammalian presenilins (PS-1 and PS-2) are polytopic transmembrane proteins that appear to function as aspartyl proteases within a multiprotein, γ-secretase complex (Wolfe and Kopan, 2004). Presenilins provide the active site of the proteolytic activity. This last cleavage releases the Notch intracellular domain, initiating CBF-1-mediated signaling. Thus, pharmacologic intervention that inhibits the activity of the γ-secretase proteases can potentially inhibit tumor growth in Notch-dependent cancer. Interestingly, at the same time that presenilins were shown to be essential for Notch signaling, they were discovered as susceptibility loci for Alzheimer's disease (Levitan and Greenwald, 1995). The pathogenesis of Alzheimer's disease is believed to be the accumulation of amyloid β-peptide (Aβ), which is derived from proteolytic processing of the β-amyloid precursor protein (APP) by β- and γ-secretases. Since inhibition of the Notch3 pathway using the DN construct resulted in the loss of the tumor phenotype, the inventors wanted to examine the effect of γ-secretase inhibitors on tumor growth. The inventors treated HCC2429 cells with GSI (Gamma Secretase Inhibitor), a commercially available γ-secretase inhibitor (Calbiochem). The inventors observed that GSI inhibits tumor proliferation with an IC50 of about 1 μM in comparison to DMSO (
FIG. 7A ). However, in the presence of low serum the inhibition increases almost 1 log. This finding is similar to what was observed when the DN construct was used in HCC2429. This observation suggests that the antiproliferative activity observed is Notch-dependent. The inventors also observed inhibition of proliferation in other lung cancer cell lines expressing Notch3 (data not shown). Biochemically, treatment with these inhibitors also resulted in the loss of activated Notch3, as measured by the levels of the N3ICD (FIG. 7B ). Stable expression of Notch3 siRNA also leads to loss of sensitivity to γ-secretase inhibition (FIGS. 7C , 7D). - To assess the effect of pharmacologically inhibiting Notch activation in vivo, the inventors treated subcutaneous xenografts with MRK003, a γ-secretase inhibitor from Merck, Inc. & Co. Based on the manufacturer's recommended dose, the mice were treated with 100 mg/kg orally for 3 consecutives days per week once the tumors were palpable. At the end of a 2-week treatment, the animals were then euthanized, and the tumors were harvested. A reduction of tumor size was seen in animals treated with the inhibitor, with a concomitant reduction in activated Notch3 (N3ICD) (
FIGS. 8A-D ). - Notch3 Inhibition Induces Apoptosis. One hallmark of cancer is resistance to apoptosis. Genetic and pharmacologic inhibition of the Notch pathway in other tumors has been shown to induce apoptosis (Qin et al., 2004; Curry et al., 2005; Shelly et al., 1999). The inventors examined whether inhibiting the Notch3 pathway can induce apoptosis. The inventors noted that in the HCC2429 clone expressing the dominant-negative receptor, the inhibition of the Notch3 pathway appears to render the tumor cells sensitive to apoptosis in the presence of serum starvation compared to vector control (
FIG. 9A ). One mechanism of apoptosis induction is the down-regulation of Akt phosphorylation.FIG. 9B shows that the level of phosphorylated Akt does indeed decrease when the DN Notch3 construct is used. The inventors also demonstrated that inhibition of Notch3 using siRNA results in the down-regulation of Bcl-xL (24 and 48 hours) and enhances the upregulation of cleaved PARP (48 hours), suggesting that Notch3 plays and important role in tumor survival (FIG. 9C ). - Notch3 Crosstalks with the MAPK Pathway. In mammals, Notch receptors signal primarily by binding to CBF-1 and related transcription co-activators. However, as mentioned previously, the Notch and EGF/MAPK pathways are known to interact in developing vertebrates and invertebrates. Thus, since the MAPK pathway, and in particular the ERK subfamily, also plays a prominent role in cellular response to growth factors, and is often altered in cancer, The inventors examined whether Notch3 alters ERK signaling in lung cancer cells. Using the DN receptor, they showed that inhibiting Notch3 downregulates MAPK, and conversely, that MAPK is upregulated when the cells were transfected with activated Notch3 intracellular domain (
FIGS. 10A , 10B). Moreover, lower levels of activated MAPK (p44/p42) expression in the unstimulated DN clones and a markedly higher level of activation with growth factor induction in the VC in comparison to the DN clone suggest that the disruption of the Notch3 signaling pathway renders lung cancer cells more resistant to growth-factor-dependent MAPK activation. This observation has been confirmed in two other lung cancer cell lines, H460 and H1819, transfected with the DN construct. - Interestingly, with prolonged exposure to serum (60 minutes), the HCC2429 clone expressing the dominant-negative construct shows that p44/p42 phosphorylation was attenuated significantly compared to vector control (
FIG. 10A ). Prolonged exposure to growth factors and activation of the p44/p42 cascade can induce the expression of MAPK phosphatases-1 and -2 (MKP-1/-2) as part of a negative feedback loop and result in the down-regulation of the MAPK pathway (Traverse et al., 1992; Plows et al., 2002; Haneda et al., 1999; Brondello et al., 1997). Down-regulation of MKPs or resistance to dephosphorylation by MKPs are often observed in human tumors, and Notch3 may have a role in suppressing MKP expression (Sivaraman et al., 1997; Magi-galluzzi et al., 1997; Barry et al., 2001). Recent data demonstrate that one mechanism by which Notch antagonizes EGF in developing C. elegans is the upregulation of LIP-1, a homolog of mammalian MAPK phosphatases (Berset et al., 2001). Since Drosophila and C. elegans Notch have the highest homology to human Notch1, and Notch3 appears to antagonize Notch1, it follows that Notch3 might suppress MAPK phosphatase expression in cancer cells. To test this hypothesis, the inventors quantitated the level of MKP-1 using real-time PCR following serum induction in HCC2429 stably transfected with DN and VC. Higher levels of MKP-1 were observed in the DN clones (FIG. 10C ). This finding suggests that Notch3 also modulates MAPK activation through MKP-1 transcriptional control. - EGFr Inhibitors Enhance the Anti-proliferation Effect of Notch3 Inhibition. The inventors previously showed that Notch3 expression positively correlates with EGFR expression in our tumor tissue array. The also showed that Notch3 cooperates with the MAPK pathway. Based on existing literature and their observations, the inventors hypothesized that Notch3 acts synergistically with the EGFR pathway in the promotion and the survival of lung cancer cells and that combining inhibitors of both pathways will have synergistic therapeutic value. To test this hypothesis, the inventors examined whether inhibiting the Notch3 pathway increases tumor inhibition when lung cancer cells are treated with an EGFR tyrosine kinase inhibitor, AG1478. When HCC2429 cells were maintained in EGF-supplemented media and treated with increasing doses of AG1478, the clones transfected with the DN construct showed a 40-fold increase in sensitivity to EGFR inhibitors, that is, the IC50 was reduced from 8.3 μM to 0.2 μM (
FIG. 11A ). In H460, a lung cancer cell line that has lower Notch3 expression as well as a k-Ras mutation (data not shown) and is more resistant to AG1478 (IC50 of 23.8 μM), the inhibition of the Notch3 pathway also reduces cancer cell survival by approximately two-fold (IC50 of 12.1 μM,FIG. 10A ). These data provide evidence that Notch3 activation may decrease a tumor's dependence on the EGF pathway and thus further decrease the sensitivity to EGFR tyrosine kinase inhibitors. Synergism can also be observed when the γ-secretase inhibitor L-685,458 is added to AG1478 (FIG. 11B ). Using the soft-agar colony assay, the inventors also demonstrate additive effects by combining MRK003, another γ-secretase inhibitor, with an EGFR inhibitor (FIGS. 12A , 12B). From a therapeutic standpoint, Notch3 is a good target for therapeutic intervention both alone and in combination with growth factor receptor inhibitors. Since about 80% of lung carcinomas express EGFR, but far fewer respond to kinase inhibition, our data suggest that adding a Notch3 inhibitor will improve the response rate in patients treated with EGFR inhibitors. - Notch3 peptides induce apoptosis, inhibit Notch3-regulated gene Hey1, and interrupt signaling through binding to ligand Jagged1.
FIG. 13 is a representative experiment showing that the peptides induce apoptosis by Annexin V staining through screening using an FMAT system. Each of 155 different peptides were assayed in quadruplicate, and only those peptides that produced a significant increase of fluorescence signal in all 4 wells were considered potentially positive or capable of inducing apoptosis. The bar graphs here reflect fluorescence counts.FIG. 14A shows HCC2429 treated with Notch3 peptides N16, N17, N102, N103, N132, with induction of apoptosis by peptides compared to control. Treatment with peptides also reduced transcription of Notch3-dependent gene Hey1 as determined by real-time RT-PCR (FIG. 14B ). Of note, N17 peptide both demonstrates highest apoptotic activity and best reduction in Hey1 transcription.FIG. 15 shows HEK cells transfected with Jagged1-HA and treated with Notch3 peptides. The peptides were then immunoprecipitated from cell lysate with streptavidin beads and immunoblotted with anti-HA antibody. This suggests that peptide induces apoptosis via binding to ligand Jagged1 and preventing activation of Notch3 receptor. - Sera from mice immunized with Notch3 recombindant protein inhibit Notch3 activation.
FIG. 16 shows an immunoblot that demonstrates that sera frommice # - Recombinant Fc-fusion Notch3 proteins inhibit Notch3 activation and induces apoptosis in vitro.
FIG. 17A shows Fc-fusion protein comprised for N16-17 and N132 sequences inhibits Notch3 activation, whileFIG. 17B shows that purified recombinant N16-17-Fc protein induces apoptosis as compared to control and Fc control after 40 hrs treatment. This study further supports the hypothesis that these regions of Notch3 are important for ligand interaction, and that disruption of this interaction using decoy recombinant receptor can inhibit Notch3 activation. - All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
- The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:
- U.S. Pat. No. 4,196,265
- U.S. Pat. No. 4,554,101
- U.S. Pat. No. 4,683,202
- U.S. Pat. No. 4,684,611
- U.S. Pat. No. 4,879,236
- U.S. Pat. No. 4,952,500
- U.S. Pat. No. 5,217,879
- U.S. Pat. No. 5,302,523
- U.S. Pat. No. 5,322,783
- U.S. Pat. No. 5,384,253
- U.S. Pat. No. 5,464,765
- U.S. Pat. No. 5,506,138
- U.S. Pat. No. 5,538,877
- U.S. Pat. No. 5,538,880
- U.S. Pat. No. 5,550,318
- U.S. Pat. No. 5,563,055
- U.S. Pat. No. 5,580,859
- U.S. Pat. No. 5,589,466
- U.S. Pat. No. 5,610,042
- U.S. Pat. No. 5,656,610
- U.S. Pat. No. 5,670,488
- U.S. Pat. No. 5,702,932
- U.S. Pat. No. 5,736,524
- U.S. Pat. No. 5,739,018
- U.S. Pat. No. 5,780,448
- U.S. Pat. No. 5,789,215,
- U.S. Pat. No. 5,824,544
- U.S. Pat. No. 5,830,725
- U.S. Pat. No. 5,849,304
- U.S. Pat. No. 5,851,826
- U.S. Pat. No. 5,858,744
- U.S. Pat. No. 5,871,982
- U.S. Pat. No. 5,871,983
- U.S. Pat. No. 5,871,986
- U.S. Pat. No. 5,879,934
- U.S. Pat. No. 5,888,502
- U.S. Pat. No. 5,925,565
- U.S. Pat. No. 5,928,906
- U.S. Pat. No. 5,932,210
- U.S. Pat. No. 5,935,819
- U.S. Pat. No. 5,945,100
- U.S. Pat. No. 5,955,331
- U.S. Pat. No. 5,981,274
- U.S. Pat. No. 5,994,136
- U.S. Pat. No. 5,994,624
- U.S. Pat. No. 6,013,516
- Ahmad et al., Dev. Biol., 194:86-98, 1998.
- Almendro et al., J. Immunol., 157(12):5411-5421, 1996.
- Alves da Costa et al., J. Neurochem., 90:800-806, 2004.
- Amado and Chen, Science, 285(5428):674-676, 1999.
- Angel et al., Cell, 49:729, 1987b.
- Angel et al., Mol. Cell. Biol., 7:2256, 1987a.
- Armentano et al., Proc. Natl. Acad. Sci. USA, 87(16):6141-6145, 1990.
- Artavanis-Tsakonas et al., Science, 284:770-776, 1999.
- Atchison and Perry, Cell, 46:253, 1986.
- Atchison and Perry, Cell, 48:121, 1987.
- Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, New York, 1994.
- Banerji et al., Cell, 27(2 Pt 1):299-308, 1981.
- Banerji et al., Cell, 33(3):729-740, 1983.
- Barany and Merrifield, In: The Peptides, Gross and Meienhofer (Eds.), Academic Press, NY, 1-284, 1979.
- Barry et al., J. Biol. Chem., 276:15537-15546, 2001.
- Bates, Mol. Biotechnol., 2(2):135-145, 1994.
- Batra et al., Am. J. Respir. Cell Mol. Biol., 21(2):238-245, 1999.
- Battraw and Hall, Theor. App. Genet., 82(2):161-168, 1991.
- Beatus and Lendahl, J. Neurosci. Res., 54:125-136, 1998.
- Bellavia et al., Embo. J, 19:3337-3348, 2000.
- Bellavia et al., Proc. Natl. Acad. Sci. USA, 99:3788-3793, 2002.
- Berkhout et al., Cell, 59:273-282, 1989.
- Berset et al., Science, 291:1055-1058, 2001.
- Bett et al., J. Virololgy, 67(10):5911-5921, 1993.
- Bhattacharjee et al., J. Plant Bioch. Biotech., 6(2):69-73. 1997.
- Bilbao et al., Transplant Proc., 31(1-2):792-793, 1999.
- Blackwell et al., Arch. Otolaryngol. Head. Neck Surg., 125(8):856-863, 1999.
- Blanar et al, EMBO J., 8:1139, 1989.
- Blomer et al., J. Virol., 71(9):6641-6649, 1997.
- Bodine and Ley, EMBO J., 6:2997, 1987.
- Boshart et al., Cell, 41:521, 1985.
- Bosze et al., EMBO J., 5(7):1615-1623, 1986.
- Braddock et al., Cell, 58:269, 1989.
- Brondello et al., J. Biol. Chem., 272:1368-1376, 1997.
- Bulla and Siddiqui, J. Virol., 62:1437, 1986.
- Callahan and Egan, J. Mammary Gland Biol. Neoplasia., 9:145-163, 2004.
- Campbell and Villarreal, Mol. Cell. Biol., 8:1993, 1988.
- Campbell et al., Am. Rev. Respir. Dis., 130(3):417-423, 1984.
- Campere and Tilghman, Genes and Dev., 3:537, 1989.
- Campo et al., Nature, 303:77, 1983.
- Campos et al., Circ. Res., 91:999-1006, 2002.
- Capaldi et al., Biochem. Biophys. Res. Comm., 76:425, 1977.
- Caplen et al., Gene Ther., 6(3):454-459, 1999.
- Carbonelli et al., FEMS Microbiol Lett., 177(1):75-82, 1999.
- Case et al., Proc. Natl. Acad. Sci. USA, 96(6):2988-2893, 1999.
- Celander and Haseltine, J. Virology, 61:269, 1987.
- Celander et al., J. Virology, 62:1314, 1988.
- Chandler et al., Cell, 33:489, 1983.
- Chandler et al., Proc. Natl. Acad. Sci. USA, 94(8):3596-601, 1997.
- Chang et al., Mol. Cell. Biol., 9:2153, 1989.
- Chatterjee et al., Proc. Natl. Acad. Sci. USA, 86:9114, 1989.
- Chen and Okayama, Mol. Cell. Biol. 7:2745-2752, 1987.
- Chen et al., Genes Dev., 10:2438-2451, 1996.
- Chillon et al., J. Virol., 73(3):2537-2540, 1999.
- Christou et al., Proc. Natl. Acad. Sci. USA, 84(12):3962-3966, 1987.
- Clay et al., Pathol. Oncol. Res., 5(1):3-15, 1999.
- Cocea, Biotechniques, 23(5):814-816, 1997.
- Coffey et al., Science, 282(5392):1332-1334, 1998.
- Cohen et al., J. Cell. Physiol., 5:75, 1987.
- Cook et al., Cell, 27:487-496, 1981.
- Costa et al., Mol. Cell. Biol., 8:81, 1988.
- Cripe et al., EMBO J., 6:3745, 1987.
- Culotta and Hamer, Mol. Cell. Biol., 9:1376, 1989.
- Culver et al., Science, 256(5063):1550-1552, 1992.
- Curry et al., Oncogene, 24:6333-6344, 2005.
- D'Halluin et al., Plant Cell, 4(12):1495-1505, 1992.
- Dandolo et al., J. Virology, 47:55-64, 1983.
- Dang et al., J. Natl. Cancer Inst., 92:1355-1357, 2000.
- De Villiers et al., Nature, 312(5991):242-246, 1984.
- DeLuca et al., J. Virol., 56(2):558-570, 1985.
- Deng et al., Cell, 82:675-684, 1995.
- Derby et al., Hear Res., 134(1-2):1-8, 1999.
- Deschamps et al., Science, 230:1174-1177, 1985.
- Domenga et al., Genes Dev., 18:2730-2735, 2004.
- Dorai et al., Int. J. Cancer, 82(6):846-852, 1999.
- Dovey et al., J. Neurochem., 76:173-181, 2001.
- Edbrooke et al., Mol. Cell. Biol., 9:1908, 1989.
- Ellisen et al., Cell, 66:649-661, 1991.
- Engel and Kohn, Front Biosci., 4:e26-33, 1999.
- EPO 0273085
- Faux et al., J. Neurosci., 21:5587-5596, 2001.
- Fechheimer, et al., Proc Natl. Acad. Sci. USA, 84:8463-8467, 1987.
- Feldman et al., Semin. Interv. Cardiol., 1(3):203-208, 1996.
- Feng and Holland, Nature, 334:6178, 1988.
- Feng et al., Nat. Biotechnol., 15(9):866-870, 1997.
- Firak and Subramanian, Mol. Cell. Biol., 6:3667, 1986.
- Fisher et al., Virology, 217(1):11-22, 1996.
- Fitzgerald et al., Oncogene, 19:4191-4198, 2000.
- Foder et al., Science, 251:767-773, 1991.
- Foecking and Hofstetter, Gene, 45(1):101-105, 1986.
- Forster and Symons, Cell, 49:211-220, 1987.
- Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979.
- Freifelder, In: Physical Biochemistry Applications to Biochemistry and Molecular Biology, 2nd Ed. Wm. Freeman and Co., NY, 1982.
- Frohman, In: PCR Protocols: A Guide To Methods And Applications, Academic Press, N.Y., 1990.
- Fujita et al., Cell, 49:357, 1987.
- Fujiwara and Tanaka, Nippon Geka Gakkai Zasshi, 99(7):463-468, 1998.
- Garoff and Li, Curr. Opin. Biotechnol., 9(5):464-469, 1998.
- Gamido et al., J. Neurovirol., 5(3):280-288, 1999.
- Gefter et al., Somatic Cell Genet., 3:231-236, 1977.
- Gerlach et al., Nature (London), 328:802-805, 1987.
- Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu and Wu (Eds.), Marcel Dekker, New York, 87-104, 1991.
- Gilles et al., Cell, 33:717, 1983.
- Glasser et al., Am. J. Physiol., 261:L349-356, 1991.
- Gloss et al., EMBO J., 6:3735, 1987.
- Gnant et al., Cancer Res., 59(14):3396-403, 1999.
- Gnant et al., J. Natl. Cancer Inst., 91(20):1744-1750, 1999.
- Godbout et al., Mol. Cell. Biol., 8:1169, 1988.
- Goding, In: Monoclonal Antibodies: Principles and Practice, 2d ed., Orlando, Fla., Academic Press, 60-61, 65-66, 71-74, 1986.
- Goodbourn and Maniatis, Proc. Natl. Acad. Sci. USA, 85:1447, 1988.
- Goodbourn et al., Cell, 45:601, 1986.
- Gopal, Mol. Cell. Biol., 5:1188-1190, 1985.
- Graham and Prevec Mol. Biotechnol., 3(3):207-220, 1995.
- Graham and Van Der Eb, Virology 52:456-467, 1973
- Greene et al., Immunology Today, 10:272, 1989
- Grosschedl and Baltimore, Cell, 41:885, 1985.
- Hacia et al., Nature Genet., 14:441-449, 1996.
- Haecker et al., Hum. Gene Ther., 7(15):1907-1914, 1996.
- Han et al., Euro. J Surgical Oncology, 25:194-198, 1999.
- Haneda et al., Eur. J. Pharmacol., 365:1-7, 1999.
- Harland and Weintraub, J. Cell Biol., 101: 1094-1099, 1985.
- Harlow and Lane, In: Antibodies: A laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1988.
- Haruki et al., Cancer Res., 65:3555-3561, 2005.
- Haslinger and Karin, Proc. Natl. Acad. Sci. USA, 82:8572, 1985.
- Hauber and Cullen, J. Virology, 62:673, 1988.
- Hayashi et al., Neurosci. Lett., 267(1):37-40, 1999.
- He et al., Plant Cell Reports, 14 (2-3):192-196, 1994.
- Hen et al., Nature, 321:249, 1986.
- Hensel et al., Lymphokine Res., 8:347, 1989.
- Hermens and Verhaagen, Prog. Neurobiol., 55(4):399-432, 1998.
- Herr and Clarke, Cell, 45:461, 1986.
- Hirochika et al., J. Virol., 61:2599, 1987.
- Hirsch et al., Br. J. Cancer, 86:1449-1456, 2002.
- Hirsch et al., Mol. Cell. Biol., 10:1959, 1990.
- Hogan et al., In: Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1994.
- Holbrook et al., Virology, 157:211, 1987.
- Holley, Nature, 258:487-490, 1975.
- Holzer et al., Virology, 253(1):107-114, 1999.
- Horlick and Benfield, Mol. Cell. Biol., 9:2396, 1989.
- Hou and Lin, Plant Physiology, 111:166, 1996.
- Howard et al., Ann. NY Acad. Sci., 880:352-365, 1999.
- Hrabe de Angelis et al., Nature, 386:717-721, 1997.
- Huang et al., Cell, 27:245, 1981.
- Huard et al., Neuromuscul Disord., 7(5):299-313, 1997.
- Hug et al., Mol. Cell. Biol., 8:3065, 1988.
- Hwang et al., Mol. Cell. Biol., 10:585, 1990.
- Imagawa et al., Cell, 51:251, 1987.
- Imai et al., J. Virol., 72(5):4371-4378, 1998.
- Imbra and Karin, Nature, 323:555, 1986.
- Imler et al., Mol. Cell. Biol., 7:2558, 1987.
- Imperiale and Nevins, Mol. Cell. Biol., 4:875, 1984.
- Innis et al., Proc Natl Acad Sci USA, 85(24):9436-9440, 1988.
- Irie et al., Antisense Nucleic Acid Drug Dev., 9(4):341-349, 1999.
- Jakobovits et al., Mol. Cell. Biol., 8:2555, 1988.
- Jameel and Siddiqui, Mol. Cell. Biol., 6:710, 1986.
- Jaynes et al., Mol. Cell. Biol., 8:62, 1988.
- Jemal et al., CA Cancer J. Clin., 55:10-30, 2005.
- Jhappan et al., Genes Dev., 6:345-355, 1992.
- Johnson et al., IN: Biotechnology And Pharmacy, Pezzuto et al., (Eds.), Chapman and Hall, New York, 1993.
- Johnson et al., Mol. Cell. Biol., 9:3393, 1989.
- Johnston and Edgar, Nature, 394:82-84, 1998.
- Johnston et al., J. Virol., 73(6):4991-5000, 1999.
- Joutel et al., J. Clin. Invest., 105:597-605, 2000.
- Joyce, Nature, 338:217-244, 1989.
- Kadesch and Berg, Mol. Cell. Biol., 6:2593, 1986.
- Kaeppler et al., Plant Cell Reports, 9:415-418, 1990.
- Kaneda et al., Science, 243:375-378, 1989.
- Karin et al., Mol. Cell. Biol., 7:606, 1987.
- Katinka et al., Cell, 20:393, 1980.
- Kato et al, J. Biol. Chem., 266(6):3361-3364, 1991.
- Kaufman et al., Surv. Opthalmol., 43 Suppl 1: S91-97, 1999.
- Kawamoto et al., Mol. Cell. Biol., 8:267, 1988.
- Kay, Haemophilia, 4(4):389-392, 1998.
- Kiledjian et al., Mol. Cell. Biol., 8:145, 1988.
- Kim and Cech, Proc. Natl. Acad. Sci. USA, 84:8788-8792, 1987.
- Klamut et al., Mol. Cell. Biol., 10: 193, 1990.
- Klimatcheva et al., Front Biosci., 4:D481-96, 1999.
- Koch et al., Mol. Cell. Biol., 9:303, 1989.
- Kohler and Milstein, Eur. J. Immunol., 6:511-519, 1976.
- Kohler and Milstein, Nature, 256:495-497, 1975.
- Kohut et al., Am. J. Physiol., 275(6 Pt 1):L1089-94, 1998.
- Kooby et al., FASEB J, 13(11):1325-1334, 1999.
- Kornberg, In: DNA Replication, W. H. Freeman and Company, New York, 1992.
- Kraus et al., FEBS Lett., 428(3):165-170, 1998.
- Krebs et al., Genes Dev., 14:1343-1352, 2000.
- Kriegler and Botchan, In: Eukaryotic Viral Vectors, Gluzman (Ed.), Cold Spring Harbor: Cold Spring Harbor Laboratory, NY, 1982.
- Kriegler and Botchan, Mol. Cell. Biol., 3:325, 1983.
- Kriegler et al., Cell, 38:483, 1984.
- Kriegler et al., Cell, 53:45, 1988.
- Krisky et al., Gene Ther, 5(11):1517-1530, 1998a.
- Krisky et al., Gene Ther., 5(12):1593-1603, 1998b.
- Kuhl et al., Cell, 50:1057, 1987.
- Kunz et al., Nucl. Acids Res., 17:1121, 1989.
- Kwoh et al., Proc. Natl. Acad. Sci. USA, 86(4):1173-1177, 1989.
- Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982.
- Lachmann and Efstathiou, Clin. Sci. (Colch), 96(6):533-541, 1999.
- Lanford et al., Nat. Genet., 21:289-292, 1999.
- Lardelli et al., Mech Dev., 59:177-190, 1996.
- Lareyre et al., J. Biol. Chem., 274(12):8282-8290, 1999.
- Larsen et al., Proc Natl. Acad. Sci. USA., 83:8283, 1986.
- Laspia et al., Cell, 59:283, 1989.
- Latimer et al., Mol. Cell. Biol., 10:760, 1990.
- Lazzeri, Methods Mol. Biol., 49:95-106, 1995.
- Lee et al., J. Auton. Nerv. Syst., 74(2-3):86-90, 1997.
- Lee et al., Korean J. Genet., 11(2):65-72, 1989.
- Lee et al., Nature, 294:228, 1981.
- Lee et al., Nature, 329(6140):642-645, 1987.
- Lee et al., Nucleic Acids Res., 12:4191-206, 1984.
- Leibowitz et al., Diabetes, 48(4):745-753, 1999.
- Leonhardt et al., J. Cell Biol., 149:271-280, 2000.
- Lesch, Biol. Psychiatry, 45(3):247-253, 1999.
- Levenson et al., Human Gene Therapy, 9:1233-1236, 1998.
- Levinson et al., Nature, 295:79, 1982.
- Levitan and Greenwald, Nature, 377:351-354, 1995.
- Li et al., Science, 275:1943-1947, 1997.
- Liang and Pardee, Nature Reviews Cancer, 3:869-876, 2003.
- Liang, Biotechniques, 33:338-346, 2002.
- Lin et al., Mol. Cell. Biol., 10:850, 1990.
- Linggi et al., Oncogene, 25:160-163, 2006.
- Lu et al., Clin. Cancer Res., 10:3291-3300, 2004.
- Lundstrom, J. Recept. Signal Transduct. Res., 19(1-4):673-686, 1999.
- Luria et al., EMBO J., 6:3307, 1987.
- Lusky and Botchan, Proc. Natl. Acad. Sci. USA, 83:3609, 1986.
- Lusky et al., Mol. Cell. Biol., 3:1108, 1983.
- Ma et al., Hematol. Oncol., 17:91-105, 1997.
- Macejak and Sarnow, Nature, 353:90-94, 1991.
- Magi-Galluzzi et al., Lab Invest., 76:37-51, 1997.
- Majors and Varmus, Proc. Natl. Acad. Sci. USA, 80:5866, 1983.
- Marienfeld et al., Gene Ther., 6(6): 1101-1113, 1999.
- Mastrangelo et al., Cancer Gene Ther., 6(5):409-422 1999.
- McNeall et al., Gene, 76:81, 1989.
- Meert et al., Eur. Respir. J., 20:975-981, 2002.
- Merrifield, Science, 232(4748):341-347 1986.
- Michel and Westhof, J. Mol. Biol., 216:585-610, 1990.
- Miksicek et al., Cell, 46:203, 1986.
- Miller et al., Methods Enzymol., 217:581-599, 1993.
- Mitsiadis et al. Dev. Biol., 204:420-431, 1998.
- Miyamoto et al., Cancer Cell, 3:565-576, 2003.
- Miyatake et al., Gene Ther., 6(4):564-572, 1999.
- Moldawer et al., Shock, 12(2):83-101, 1999.
- Mordacq and Linzer, Genes and Dev., 3:760, 1989.
- Moreau et al., Nucl. Acids Res., 9:6047, 1981.
- Moriuchi et al., Cancer Res., 58(24):5731-5737, 1998.
- Morrison et al., J. Gen. Virol., 78(Pt 4):873-878, 1997.
- Muesing et al., Cell, 48:691, 1987.
- Mumm and Kopan, Dev. Biol., 228:151-165, 2000.
- Nahle et al., Nat. Cell Biol., 4:859-864, 2002.
- Naldini et al., Proc. Natl. Acad. Sci. USA, 93(21):11382-11388, 1996.
- Neumann et al., Proc. Natl. Acad. Sci. USA, 96(16):9345-9350, 1999.
- Ng et al., Nuc. Acids Res., 17:601, 1989.
- Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.
- Nicolau et al., Methods Enzymol., 149:157-176, 1987
- Nomoto et al., Gene, 236(2):259-271, 1999.
- Ohara et al., Proc. Natl. Acad. Sci. USA, 86:5673-5677, 1989.
- Omirulleh et al., Plant Mol. Biol., 21(3):415-28, 1993.
- Ondek et al., EMBO J., 6:1017, 1987.
- Ornitz et al., Mol. Cell. Biol., 7:3466, 1987.
- Oswald et al., Mol. Cell. Biol., 18:2077-2088, 1998.
- Palmiter et al., Nature, 300:611, 1982.
- Paris et al., Eur. J. Pharmacol., 514:1-15, 2005.
- Parks et al., J. Virol., 71(4):3293-8, 1997.
- PCT Appln. WO 9217598
- PCT Appln. WO 94/09699
- PCT Appln. WO 95/06128
- Pear et al., J. Exp. Med., 183:2283-2291, 1996.
- Pease et al., Proc. Natl. Acad. Sci. USA, 91:5022-5026, 1994.
- Pech et al., Mol. Cell. Biol., 9:396, 1989.
- Pelletier and Sonenberg, Nature, 334:320-325, 1988.
- Pelletier et al., Cancer Res., 66:3681-3687, 2006.
- Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994.
- Perez-Stable and Constantini, Mol. Cell. Biol., 10:1116, 1990.
- Petrof, Eur. Respir. J., 11(2):492-497, 1998.
- Picard and Schaffner, Nature, 307:83, 1984.
- Pignon J et al., Hum. Mutat., 3(2):126-132, 1994.
- Pinkert et al., Genes and Dev., 1:268, 1987.
- Plows et al., Biochem J., 362:305-315, 2002.
- Polyak et al., Genes Dev., 10:1945-1952, 1996.
- Ponta et al., Proc. Natl. Acad. Sci. USA, 82:1020, 1985.
- Porton et al., Mol. Cell. Biol., 10: 1076, 1990.
- Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985.
- Potter et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165, 1984.
- Purow et al., Cancer Res., 65:2353-2363, 2005.
- Qin et al., Mol. Cancer. Ther., 3:895-902, 2004.
- Queen and Baltimore, Cell, 35:741, 1983.
- Quinn et al., Mol. Cell. Biol., 9:4713, 1989.
- Rabinovitch et al., Diabetes, 48(6):1223-1229, 1999.
- Rebay et al., Cell, 74:319-329, 1993.
- Reddy et al., J. Virol., 72(2):1394-1402, 1998.
- Redondo et al., Science, 247:1225, 1990.
- Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.
- Reisman and Rotter, Mol. Cell. Biol., 9:3571, 1989.
- Remington's Pharmaceutical Sciences, 15th Ed., 33:624-652, 1990.
- Remington's Pharmaceutical Sciences, 15th Ed., 1035-1038 and 1570-1580, 1990.
- Resendez Jr. et al., Mol. Cell. Biol., 8:4579, 1988.
- Rhodes et al., Methods Mol. Biol., 55:121-131, 1995.
- Ripe et al., Mol. Cell. Biol., 9:2224, 1989.
- Rippe et al., Mol. Cell. Biol., 10:689-695, 1990.
- Rittling et al., Nuc. Acids Res., 17:1619, 1989.
- Robbins and Ghivizzani, Pharmacol. Ther., 80(1):35-47, 1998.
- Robbins et al., Proc. Natl. Acad. Sci. USA, 95(17):10182-10187 1998.
- Robbins et al., Trends Biotechnol., 16(1):35-40, 1998.
- Robey et al., Cell, 87:483-492, 1996.
- Rodenhuis et al., J. Clin. Oncol., 15:285-291, 1997.
- Rohn et al., J. Virol., 70:8071-8080, 1996.
- Rosen et al., Cell, 41:813, 1988.
- Sakai et al., Genes and Dev., 2:1144, 1988.
- Sambrook et al., In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1(7):7.19-17.29, 1989.
- Santagata et al., Cancer Res., 64:6854-6857, 2004.
- Sarver et al., Science, 247:1222-1225, 1990.
- Satake et al., J. Virology, 62:970, 1988.
- Sawai et al., Mol. Genet. Metab., 67(1):36-42, 1999.
- Scanlon et al., Proc. Natl. Acad. Sci. USA, 88:10591-10595, 1991.
- Schaffner et al., J. Mol. Biol., 201:81, 1988.
- Searle et al., Mol. Cell. Biol., 5:1480, 1985.
- Sharp and Marciniak, Cell, 59:229, 1989.
- Shaul and Ben-Levy, EMBO J., 6:1913, 1987.
- Shelly et al., J. Cell Biochem., 73:164-175, 1999.
- Sherman et al., Mol. Cell. Biol., 9:50, 1989.
- Shimizu et al., Mol. Cell. Biol., 20:6913-6922, 2000.
- Shoemaker et al., Nature Genetics, 14:450-456, 1996.
- Sivaraman et al., J. Clin. Invest., 99:1478-1483, 1997.
- Sleigh and Lockett, J. EMBO, 4:3831, 1985.
- Small et al., J. Biol. Chem., 278:16405-16413, 2003.
- Smith, Arch. Neurol., 55(8):1061-1064, 1998.
- Spalholz et al., Cell, 42:183, 1985.
- Spandau and Lee, J. Virology, 62:427, 1988.
- Spandidos and Wilkie, EMBO J., 2:1193, 1983.
- Stambolic et al., Mol. Cell, 8:317-325, 2001.
- Steck et al., Nat. Genet., 15:356-362, 1997.
- Stein et al., J. Biol. Chem., 279:48930-48940, 2004.
- Stephens and Hentschel, Biochem. J, 248:1, 1987.
- Stewart and Young, In: Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., 1984.
- Stewart et al., Gene Ther., 6(3):350-363, 1999.
- Stuart et al., Nature, 317:828, 1985.
- Sullivan and Peterlin, Mol. Cell. Biol., 7:3315, 1987.
- Sundaram, Genes Dev., 19:1825-1839, 2005.
- Suzuki et al., Biochem. Biophys. Res. Commun., 252(3):686-690, 1998.
- Swartzendruber and Lehman, J. Cell. Physiology, 85:179, 1975.
- Sweeney et al., Faseb. J., 18:1421-1423, 2004.
- Taichman et al., Dev. Dyn., 225:166-175, 2002.
- Takebe et al., Mol. Cell. Biol., 8:466, 1988.
- Tam et al., J. Am. Chem. Soc., 105:6442, 1983.
- Tanaka et al., Oncogene, 8:2253-2258, 1993.
- Taniura et al., J. Biol. Chem., 274:16242-16248, 1999.
- Tavernier et al., Nature, 301:634, 1983.
- Taylor and Kingston, Mol. Cell. Biol., 10:165, 1990a.
- Taylor and Kingston, Mol. Cell. Biol, 10:176, 1990b.
- Taylor and Stark, Oncogene, 20:1803-1815, 2001.
- Taylor et al., J. Biol. Chem., 264:15160, 1989.
- Thiesen et al., J. Virology, 62:614, 1988.
- Timiryasova et al., Int. J. Oncol., 14(5):845-854, 1999.
- Timiryasova et al., Oncol. Res.; 11(3):133-144, 1999.
- Traverse et al., Biochem. J, 288 (Pt 2):351-355, 1992.
- Treisman, Cell, 42:889, 1985.
- Tronche et al., Mol. Biol. Med., 7:173, 1990.
- Trudel and Constantini, Genes and Dev., 6:954, 1987.
- Tsukada et al., Plant Cell Physiol., 30(4)599-604, 1989.
- Tsumaki et al., J. Biol. Chem., 273(36):22861-22864, 1998.
- Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718, 1986.
- Tyndell et al., Nuc. Acids. Res., 9:6231, 1981.
- Vanderkwaak et al., Gynecol. Oncol., 74(2):227-234, 1999.
- Vannice and Levinson, J. Virology, 62:1305, 1988.
- Vasseur et al., Proc Natl. Acad. Sci. USA, 77:1068, 1980.
- Vogelstein et al., Nature, 408(6810):307-310, 2000.
- Vogelstein, Nature, 348(6303):681-682, 1990.
- Vousden and Lu, Nat. Rev. Cancer, 2:594-604, 2002.
- Vousden and Prives, Cell, 120:7-10, 2005.
- Wagner et al., Science, 260:1510-1513, 1990.
- Walker et al., Nucleic Acids Res., 20(7):1691-1696, 1992.
- Wang and Calame, Cell, 47:241, 1986.
- Wang et al., Gynecol. Oncol., 71(2):278-287, 1998.
- Wang et al., J. Biol. Chem., 277:23165-23171, 2002.
- Weber et al., Cell, 36:983, 1984.
- Weihl et al., Neurosurgery, 44(2):239-252, 1999.
- Weinberg et al., Biochemistry, 28:8263-8269, 1989.
- Weinberger et al., Mol. Cell. Biol., 8:988, 1984.
- Weng et al., Science, 306:269-271, 2004.
- White et al., J. Virol., 73(4):2832-28340, 1999.
- Williams et al., Blood, 107(3):931-939, 2006.
- Wilson, J. Clin. Invest., 98(11):2435, 1996.
- Winoto and Baltimore, Cell, 59:649, 1989.
- Wolfe and Kopan, Science, 305:1119-1123, 2004.
- Wong et al., Gene, 10:87-94, 1980.
- Wu and Wallace, Genomics, 4:560-569, 1989.
- Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993.
- Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987.
- Wu et al., Biochem. Biophys. Res. Commun., 233(1):221-226, 1997.
- Wu, Chung Hua Min Kuo Hsiao Erh Ko I Hsueh Hui Tsa Chih, 39(5):297-300, 1998.
- Xue et al., Hum Mol Genet., 8:723-730, 1999.
- Yamada et al., Proc. Natl. Acad. Sci. USA, 96(7):4078-4083, 1999.
- Yang and Liang, Mol. Biotechnol., 3:197-208, 2004.
- Yeung et al., Gene Ther., 6(9):1536-1544, 1999.
- Yoo et al., Science, 303:663-666, 2004.
- Yoon et al., J. Gastrointest. Surg., 3(1):34-48, 1999.
- Yu and Zhang, Biochem. Biophys. Res. Commun., 331:851-858, 2005.
- Yu et al., Proc. Natl. Acad. Sci. USA, 100:1931-1936, 2003.
- Yu et al., Proc. Natl. Acad. Sci. USA, 96:14517-14522, 1999.
- Yutzey et al., Mol. Cell. Biol., 9:1397, 1989.
- Zeng et al., Cancer Cell, 8:13-23, 2005.
- Zhao-Emonet et al., Biochim. Biophys. Acta, 1442(2-3):109-119, 1998.
- Zheng et al., J. Gen. Virol., 80(Pt 7):1735-1742, 1999.
- Zhou et al., Exp. Hematol, 21:928-933, 1993.
- Zufferey et al., Nat. Biotechnol., 15(9):871-875, 1997.
Claims (6)
1-31. (canceled)
32. An isolated and purified antibody that binds to an epitope comprising a sequence selected from the group consisting of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8).
33. A method of inhibiting Notch3 receptor signaling comprising contacting a cell expressing Notch3 with an antibody that binds to an epitope comprising a sequence selected from the group consisting of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8).
34. A method of treating a subject having a Notch3-expressing cancer comprising administering to said subject an antibody that binds to an epitope comprising a sequence selected from the group consisting of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8).
35. A pharmaceutical formulation comprising an antibody that binds to an epitope comprising a sequence selected from the group consisting of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8).
36. (canceled)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/896,439 US20110059096A1 (en) | 2007-09-14 | 2010-10-01 | Targeting of Notch3 Receptor Function for Cancer Therapy |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US97258407P | 2007-09-14 | 2007-09-14 | |
US12/208,875 US7807630B2 (en) | 2007-09-14 | 2008-09-11 | Targeting of Notch3 receptor function for cancer therapy |
US12/896,439 US20110059096A1 (en) | 2007-09-14 | 2010-10-01 | Targeting of Notch3 Receptor Function for Cancer Therapy |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/208,875 Division US7807630B2 (en) | 2007-09-14 | 2008-09-11 | Targeting of Notch3 receptor function for cancer therapy |
Publications (1)
Publication Number | Publication Date |
---|---|
US20110059096A1 true US20110059096A1 (en) | 2011-03-10 |
Family
ID=39862912
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/208,875 Expired - Fee Related US7807630B2 (en) | 2007-09-14 | 2008-09-11 | Targeting of Notch3 receptor function for cancer therapy |
US12/896,439 Abandoned US20110059096A1 (en) | 2007-09-14 | 2010-10-01 | Targeting of Notch3 Receptor Function for Cancer Therapy |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/208,875 Expired - Fee Related US7807630B2 (en) | 2007-09-14 | 2008-09-11 | Targeting of Notch3 receptor function for cancer therapy |
Country Status (2)
Country | Link |
---|---|
US (2) | US7807630B2 (en) |
WO (1) | WO2009036167A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9974774B2 (en) | 2013-07-26 | 2018-05-22 | Race Oncology Ltd. | Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7919092B2 (en) | 2006-06-13 | 2011-04-05 | Oncomed Pharmaceuticals, Inc. | Antibodies to notch receptors |
JP5618544B2 (en) | 2007-01-24 | 2014-11-05 | オンコメッドファーマシューティカルズ インコーポレイテッド | Compositions and methods for diagnosis and treatment of cancer |
WO2010005567A2 (en) | 2008-07-08 | 2010-01-14 | Oncomed Pharmaceuticals, Inc. | Notch1 receptor binding agents and methods of use thereof |
US9132189B2 (en) | 2008-07-08 | 2015-09-15 | Oncomed Pharmaceuticals, Inc. | Notch1 binding agents and methods of use thereof |
WO2011140295A2 (en) * | 2010-05-06 | 2011-11-10 | President And Fellows Of Harvard College | Modulators of notch receptor signaling and methods of use thereof |
CN104903351A (en) | 2012-11-07 | 2015-09-09 | 辉瑞公司 | Anti-notch3 antibodies and antibody-drug conjugates |
JP2016520289A (en) * | 2013-03-15 | 2016-07-14 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Method for treating pancreatic cancer |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6537775B1 (en) * | 1996-08-01 | 2003-03-25 | Institut National De La Sante Et De La Recherche (Inserm) | Gene involved in cadasil, method of diagnosis and therapeutic application |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2028187A1 (en) * | 2000-09-08 | 2009-02-25 | Board Of Regents, The University Of Texas System | Human and mouse targeting peptides identified by phage display |
AU2002339157A1 (en) * | 2001-11-14 | 2003-05-26 | Lorantis Limited | Inhibitors of the notch signalling pathway for use in the treatment of cancer |
EP1660628A4 (en) * | 2003-08-19 | 2010-03-31 | Agos Biotech Ltd | SPLICE VARIANTS OF ErbB LIGANDS, COMPOSITIONS AND USES THEREOF |
WO2006047878A1 (en) * | 2004-11-03 | 2006-05-11 | British Columbia Cancer Agency Branch | Cancer therapeutics and methods for their use |
EP2099827B1 (en) | 2006-12-18 | 2018-11-21 | Genentech, Inc. | Antagonist anti-notch3 antibodies and their use in the prevention and treatment of notch3-related diseases |
-
2008
- 2008-09-11 WO PCT/US2008/076008 patent/WO2009036167A1/en active Application Filing
- 2008-09-11 US US12/208,875 patent/US7807630B2/en not_active Expired - Fee Related
-
2010
- 2010-10-01 US US12/896,439 patent/US20110059096A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6537775B1 (en) * | 1996-08-01 | 2003-03-25 | Institut National De La Sante Et De La Recherche (Inserm) | Gene involved in cadasil, method of diagnosis and therapeutic application |
Non-Patent Citations (4)
Title |
---|
Joutel et al., The ectodomain of the Notch3 receptor accumulates within the cerbrovasculatureof CADASIL patients, J. Clin. Invest. 105(5):597-605, 2000. * |
Lipman et al., Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and informative resources, ILAR J. 46(3):258-268, 2005. * |
R & D Systems 2005 Catalog,Minneapolis, R & D Systems, Inc., p. 455, 2005. * |
R & D Systems, Product details: Human Notch-3 affinity purified polyclonal Ab, #AF1559 [online],[retrieved 2013-02-11], Retrieved from the Internet:. * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9974774B2 (en) | 2013-07-26 | 2018-05-22 | Race Oncology Ltd. | Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof |
US9993460B2 (en) | 2013-07-26 | 2018-06-12 | Race Oncology Ltd. | Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof |
US10500192B2 (en) | 2013-07-26 | 2019-12-10 | Race Oncology Ltd. | Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof |
US10548876B2 (en) | 2013-07-26 | 2020-02-04 | Race Oncology Ltd. | Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof |
US11135201B2 (en) | 2013-07-26 | 2021-10-05 | Race Oncology Ltd. | Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof |
US11147800B2 (en) | 2013-07-26 | 2021-10-19 | Race Oncology Ltd. | Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof |
Also Published As
Publication number | Publication date |
---|---|
US20090092615A1 (en) | 2009-04-09 |
US7807630B2 (en) | 2010-10-05 |
WO2009036167A1 (en) | 2009-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11136410B2 (en) | Antibodies against the MUC1-C/extracellular domain (MUC1-C/ECD) | |
US7807630B2 (en) | Targeting of Notch3 receptor function for cancer therapy | |
US7169384B2 (en) | Tumor suppressor CAR-1 | |
US10617773B2 (en) | Antibodies against the MUC1-C/extracellular domain (MUC1-C/ECD) | |
US9140705B2 (en) | Tumor suppressor killin | |
US8153368B2 (en) | Four-jointed box (FJX1) in cancer diagnosis and treatment | |
EP3065775B1 (en) | Vh4 antibodies against gray matter neuron and astrocyte | |
US9950044B2 (en) | Treatment of pulmonary vascular remodeling with neprilysin | |
US20130101664A1 (en) | Muc1 ligand traps for use in treating cancers | |
US9937250B2 (en) | Immunogenic tumor associated stromal cell antigen peptides and methods of their use | |
US20030108920A1 (en) | Tumor suppressor-like proteins that bind IGFBP2 | |
US20090263396A1 (en) | Inhibitors of t-darpp for use in combination anti-cancer therapies | |
US8716014B2 (en) | Adenovirus E1A fragments for use in anti-cancer therapies | |
US10618944B2 (en) | Tumor suppressor SALL1 as a therapeutic agent for treating cancer | |
US11518800B2 (en) | Mutations that drive VH4 antibody autoreactivity | |
US20180153978A1 (en) | TUMORS EXPRESSING IgG1 Fc INDUCE ROBUST CD8 T CELL RESPONSES | |
US20220096614A1 (en) | Peptide-induced nk cell activation | |
CA3186181A1 (en) | Antibodies against the muc1-c/extracellular domain (muc1-c/ecd) | |
KR20110011595A (en) | Tapasin augmentation for enhanced immune response | |
Rivoltini et al. | Tumor immunology: Clinical perspectives |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |