WO1998018914A1 - Recepteur tie2 soluble - Google Patents
Recepteur tie2 soluble Download PDFInfo
- Publication number
- WO1998018914A1 WO1998018914A1 PCT/US1997/019597 US9719597W WO9818914A1 WO 1998018914 A1 WO1998018914 A1 WO 1998018914A1 US 9719597 W US9719597 W US 9719597W WO 9818914 A1 WO9818914 A1 WO 9818914A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tumor
- 6his
- protein
- fusion protein
- exflk
- Prior art date
Links
- 108010090091 TIE-2 Receptor Proteins 0.000 title abstract description 3
- 102000012753 TIE-2 Receptor Human genes 0.000 title abstract description 3
- 206010028980 Neoplasm Diseases 0.000 claims description 191
- 210000004027 cell Anatomy 0.000 claims description 114
- 108090000623 proteins and genes Proteins 0.000 claims description 90
- 102000004169 proteins and genes Human genes 0.000 claims description 79
- 108020001507 fusion proteins Proteins 0.000 claims description 47
- 102000037865 fusion proteins Human genes 0.000 claims description 47
- 102000005962 receptors Human genes 0.000 claims description 34
- 108020003175 receptors Proteins 0.000 claims description 34
- 238000000034 method Methods 0.000 claims description 31
- 210000003038 endothelium Anatomy 0.000 claims description 29
- 101100481410 Mus musculus Tek gene Proteins 0.000 claims description 28
- 239000013598 vector Substances 0.000 claims description 27
- 101100481408 Danio rerio tie2 gene Proteins 0.000 claims description 26
- 230000014509 gene expression Effects 0.000 claims description 26
- 230000027455 binding Effects 0.000 claims description 22
- 230000005764 inhibitory process Effects 0.000 claims description 21
- 241000282414 Homo sapiens Species 0.000 claims description 19
- 230000012010 growth Effects 0.000 claims description 18
- 230000033115 angiogenesis Effects 0.000 claims description 17
- 108020004707 nucleic acids Proteins 0.000 claims description 15
- 102000039446 nucleic acids Human genes 0.000 claims description 15
- 150000007523 nucleic acids Chemical class 0.000 claims description 15
- 150000001875 compounds Chemical class 0.000 claims description 13
- 230000000694 effects Effects 0.000 claims description 13
- 230000015572 biosynthetic process Effects 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 11
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 239000013603 viral vector Substances 0.000 claims description 8
- 210000004899 c-terminal region Anatomy 0.000 claims description 5
- 239000000539 dimer Substances 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000002163 immunogen Effects 0.000 claims description 2
- 229920002704 polyhistidine Polymers 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 claims 1
- 239000004037 angiogenesis inhibitor Substances 0.000 abstract description 3
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 64
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 64
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 64
- 241000700605 Viruses Species 0.000 description 31
- 210000002889 endothelial cell Anatomy 0.000 description 27
- 239000000243 solution Substances 0.000 description 27
- 230000004614 tumor growth Effects 0.000 description 27
- 210000004072 lung Anatomy 0.000 description 26
- 241000700159 Rattus Species 0.000 description 25
- 239000003636 conditioned culture medium Substances 0.000 description 22
- 210000004881 tumor cell Anatomy 0.000 description 22
- 206010027476 Metastases Diseases 0.000 description 21
- 239000008188 pellet Substances 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 239000002953 phosphate buffered saline Substances 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 18
- 241000701447 unidentified baculovirus Species 0.000 description 18
- 241000699666 Mus <mouse, genus> Species 0.000 description 17
- 210000004087 cornea Anatomy 0.000 description 17
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 16
- 108091008605 VEGF receptors Proteins 0.000 description 16
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 16
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 15
- 238000001727 in vivo Methods 0.000 description 15
- 230000005747 tumor angiogenesis Effects 0.000 description 15
- 230000001404 mediated effect Effects 0.000 description 14
- 230000002792 vascular Effects 0.000 description 14
- 238000012546 transfer Methods 0.000 description 13
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 230000004044 response Effects 0.000 description 12
- 210000005166 vasculature Anatomy 0.000 description 12
- 241001529936 Murinae Species 0.000 description 11
- 230000006427 angiogenic response Effects 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 238000002513 implantation Methods 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 108010058398 Macrophage Colony-Stimulating Factor Receptor Proteins 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 10
- 230000004913 activation Effects 0.000 description 10
- 108010005774 beta-Galactosidase Proteins 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 239000006180 TBST buffer Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 230000009401 metastasis Effects 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 238000013508 migration Methods 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 241000701161 unidentified adenovirus Species 0.000 description 8
- 230000003612 virological effect Effects 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 230000000903 blocking effect Effects 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 7
- 239000007943 implant Substances 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 230000005012 migration Effects 0.000 description 7
- 230000037361 pathway Effects 0.000 description 7
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 7
- 206010006187 Breast cancer Diseases 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 208000034038 Pathologic Neovascularization Diseases 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 238000001415 gene therapy Methods 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 201000001441 melanoma Diseases 0.000 description 6
- 230000009963 pathologic angiogenesis Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- 206010027458 Metastases to lung Diseases 0.000 description 5
- 206010029113 Neovascularisation Diseases 0.000 description 5
- 108700020796 Oncogene Proteins 0.000 description 5
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 230000004087 circulation Effects 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 230000003511 endothelial effect Effects 0.000 description 5
- 238000003364 immunohistochemistry Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 230000006444 vascular growth Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 4
- 108010011170 Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Proteins 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 238000012286 ELISA Assay Methods 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- 102100028198 Macrophage colony-stimulating factor 1 receptor Human genes 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 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 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 208000036142 Viral infection Diseases 0.000 description 4
- 239000013543 active substance Substances 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000004132 cross linking Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000012737 fresh medium Substances 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 230000000644 propagated effect Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000002459 sustained effect Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 239000011547 Bouin solution Substances 0.000 description 3
- 102000000844 Cell Surface Receptors Human genes 0.000 description 3
- 108010001857 Cell Surface Receptors Proteins 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 238000000692 Student's t-test Methods 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 230000002491 angiogenic effect Effects 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 206010003246 arthritis Diseases 0.000 description 3
- 238000000376 autoradiography Methods 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 230000002526 effect on cardiovascular system Effects 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 239000000833 heterodimer Substances 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 210000004088 microvessel Anatomy 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000013391 scatchard analysis Methods 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229940104230 thymidine Drugs 0.000 description 3
- 210000003606 umbilical vein Anatomy 0.000 description 3
- 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
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 238000007809 Boyden Chamber assay Methods 0.000 description 2
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 101100481404 Danio rerio tie1 gene Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 241001135569 Human adenovirus 5 Species 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 101100481406 Mus musculus Tie1 gene Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108010039918 Polylysine Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 208000007135 Retinal Neovascularization Diseases 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 239000004809 Teflon Substances 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- 208000009956 adenocarcinoma Diseases 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000005266 casting Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 201000000159 corneal neovascularization Diseases 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000006471 dimerization reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 230000010595 endothelial cell migration Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 239000006451 grace's insect medium Substances 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000012194 insect media Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 2
- 229920000656 polylysine Polymers 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000009290 primary effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 108091006082 receptor inhibitors Proteins 0.000 description 2
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 2
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 108091008601 sVEGFR Proteins 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000001258 synovial membrane Anatomy 0.000 description 2
- 238000012385 systemic delivery Methods 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000012384 transportation and delivery Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FJQZXCPWAGYPSD-UHFFFAOYSA-N 1,3,4,6-tetrachloro-3a,6a-diphenylimidazo[4,5-d]imidazole-2,5-dione Chemical class ClN1C(=O)N(Cl)C2(C=3C=CC=CC=3)N(Cl)C(=O)N(Cl)C12C1=CC=CC=C1 FJQZXCPWAGYPSD-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 241000894006 Bacteria Species 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
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 206010013883 Dwarfism Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010041308 Endothelial Growth Factors Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101000930477 Mus musculus Albumin Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 208000003788 Neoplasm Micrometastasis Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 210000000648 angioblast Anatomy 0.000 description 1
- 238000002399 angioplasty Methods 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000003542 behavioural effect Effects 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
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000008274 breast adenocarcinoma Diseases 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000015861 cell surface binding Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 210000003683 corneal stroma Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 238000001739 density measurement Methods 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 231100001129 embryonic lethality Toxicity 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 102000058223 human VEGFA Human genes 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 108700040422 lipopolylysine Proteins 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000014399 negative regulation of angiogenesis Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 239000012536 storage buffer Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000005100 tissue tropism Effects 0.000 description 1
- 238000002723 toxicity assay Methods 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 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 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 230000007556 vascular defect Effects 0.000 description 1
- 230000006459 vascular development Effects 0.000 description 1
- 230000007998 vessel formation Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 229910000166 zirconium phosphate Inorganic materials 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- 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
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to a soluble receptor endothelium specific and to the use thereof as an antiangiogenic agent.
- Angiogenesis the formation of new capillaries from pre-existing blood vessels, is a fundamental process required for both normal embryonic development and the development of pathologic conditions such as cancer (Folkman, J. Natl. Cancer Inst. 82:4-6 (1990), Folkman et al, J. Biol. Chem. 267:10931-10934 (1992)).
- Tumor growth is an angiogenesis-dependent process that requires stimulation of new vessel growth (Viglietto et al, Oncogene 1 1 :1569- 1579 (1995), Kandel et al, Cell 66:1095-1104 (1991)).
- Tumor angiogenesis is most likely mediated by growth factors produced by tumor cells and/or by tumor infiltrating inflammatory cells such as macrophages or mast cells (Folkman et al, J. Biol. Chem. 267:10931-10934 (1992), Sunderkotter et al, J. Leukoc. Biol. 55:410-422 (1994)). These factors stimulate the proliferation, migration and morphogenesis of endothelial cells as a result of interaction with specific cell surface receptors. Although many factors likely contribute to vascular growth in tumors, VEGF (vascular endothelial growth factor) is currently the best candidate for an endogenous mediator of vascular growth (Connolly, J. Cell Biochem.
- VEGF and VEGF receptors A role for VEGF and VEGF receptors in tumor angiogenesis is supported by a growing number of reports demonstrating expression of VEGF and VEGF receptors in a number of different tumor types
- Tie2 (a.k.a. Tek) was identified (Dumont et al, Oncogene 8:1293-1301 (1993), Iwama et al, Res. Comm. 195:301 (1993)). Tie2 was expressed predominantly in endothelial cell precursors (angioblasts) and in endothelial cells participating in angiogenesis (Schnurch et al, Development 119:957-968 (1993), Dumont et al, Dev. Dynamics 203:80-92 (1995)).
- Tie2 function in transgenic mice resulted in embryonic lethality at day 8.5 due to defects in vascular development characterized by a reduction in endothelial cell number and a defect in the formation of microvessels (Sato et al, Nature 376:70-74 (1995), Dumont et al, Genes and Development 8:1897-1909 (1994)). Similar vascular defects occured following the disruption of a recently cloned Tie2 ligand (Suri et al, Cell 87:1171-1180 (1996), Davis et al, Cell 87:1161-1169 (1996)). These findings indicated that the Tie2 pathway was essential for the formation ofthe embryonic vasculature and suggested a role for Tie2 in pathologic angiogenesis including tumor neovascularization.
- the present invention relates to a endothelium specific receptor inhibitor comprising an extracellular portion (domain) of an endothelium specific receptor, essentially free of transmembrane domain sequences, and a non-endothelium specific receptor sequence that is essentially non-immunogenic and/or that does not mediate the formation of dimers or oligomers.
- a endothelium specific receptor inhibitor comprising an extracellular portion (domain) of an endothelium specific receptor, essentially free of transmembrane domain sequences, and a non-endothelium specific receptor sequence that is essentially non-immunogenic and/or that does not mediate the formation of dimers or oligomers.
- FIG. 1 A and IB Design and production of ExTek.6His. Schematic diagrams ofthe full-length Tie2 receptor and CSF-1 receptor, c-fms, together with their respective soluble extracellular domains fused at the carboxy-terminus to a 6 histidine tag (ExTek.6His and ExFms.6His) (Fig. 1 A). Recombinant baculoviruses were constructed for the production of soluble, recombinant ExTek.6His and ExFms.6His by insect cells. ExTek.6His and ExFms.6His were purified from the conditioned media of baculovirus infected SF9 cells by one step
- FIG. 3 Inhibition of tumor vascularization by ExTek.6His protein.
- Tumor vessel length density was measured as an indicator of tumor vascularization from photomicrographs of window chambers bearing 10 day old ExTek.6His treated tumors or control treated tumors.
- ExTek.6His protein did not adversely effect R3230AC tumor cell proliferation or viability.
- Cells were either maintained in the presence of ExTek.6His protein at 3 ⁇ M, which was approximately equivalent to the protein concentration used in the window chamber, or control solution for three days. Live cells were counted after suspension in PBS with 0.02%) trypan blue on each following day. Each point represents the mean of 3 experiments.
- the ExTek concentration was calibrated by using purified recombinant ExTek protein produced in Baculoviral expression vector. High level of ExTek (more than 1 mg/ml) in blood was achieved two days after viral infection. The expression was transient, which slowed diminished to baseline in about 10 days.
- FIGS 6 A and 6B Inhibition of well established primary tumor growth by AdExTek.
- the effect of AdExTek on primary tumor growth was tested in two different kind of well established murine tumors.
- Murine mammary tumor 4T1 (5x10 ⁇ cells per mouse) and melanoma F10.9 (5xl0-> cells per mouse) were implanted into the right flank of Balb/c and C57/BL mouse, respectively. After a palpable tumor was achieved (day 0), the mice were divided into two groups in each different tumor implanted animals. One group of mice were injected with control virus Ad ⁇ -gal directing the expression of ⁇ -galactosidase and the other group mice were injected with AdExTek virus iv through retro-obital sinus.
- the tumor size was measured afterward for 12 days.
- the ExTek expression was tested 2 days after viral injection.
- a significant inhibition on tumor growth was observed in AdExTek treated either 4T1 tumor group and F10.9 tumor group vs. control Ad ⁇ -gal treated animals.
- the inhibition was stronger during the first 6 days which correlated with high level expression of ExTek in the blood.
- the tumor growth inhibition decreased with the decrease of ExTek expression in blood 6 days after viral injection.
- FIGS 7A-7D AdExTek suppresses the tumor lung metastases growth.
- the inhibition of AdExTek on tumor lung metastasis was analyzed by measuring the lung weight and counting metastases on lung surface under a dissecting microscopy from both 4T1 tumor group (Figs. 7A and 7B) and F10.9 tumor group (Figs. 7C and 7D).
- lung weight in control Ad ⁇ -gal treated 4T1 tumor group (Fig. 7A) or F10.9 tumor group (Fig. C) was more than twice ofthe AdExTek treated tumor groups or normal, uninjected mouse lung. There was no significant difference in lung weight between AdExTek treated group vs. normal mouse lung.
- FIG. 8A Schematic diagrams ofthe full-length VEGF receptor, flk-1, and the full length CSF-1 receptor, c-fms, compared to the recombinant soluble extracellular domains of flk-1 (ExFlk.6His) and c-fms (ExFms. ⁇ His) fused to a 6 histidine tag at the carboxy-terminus.
- Fig. 8A Schematic diagrams ofthe full-length VEGF receptor, flk-1, and the full length CSF-1 receptor, c-fms, compared to the recombinant soluble extracellular domains of flk-1 (ExFlk.6His) and c-fms (ExFms. ⁇ His) fused to a 6 histidine tag at the carboxy-terminus.
- FIGS 9A-9C ExFlk.6His binds VEGF and inhibits VEGF mediated endothelial cell mitogenesis and migration in vitro.
- Fig. 9A Saturation binding and Scatchard analysis of ExFlk.6His binding to VEGF. Purified ExFlk.6His was radiolabelled with Na 125 I. Increasing amounts of I25 I labeled ExFlk.6His were then added to micro- wells pre-coated with VEGF (lOng/well) for 1 hour at room temperature. The wells were washed and radioactivity was detected by a r- counter. Nonspecific binding was determined by incubation of 125 I-labeled
- Fig. 9B ExFlk.6His blocked VEGF -induced thymidine incorporation in endothelial cells. Human umbilical vein endothelial cells(HUVECs) starved for 24 hours in quiescence medium were stimulated with VEGF (lOng/ml), VEGF plus ExFlk.6His (2.5 ⁇ g/ml) or VEGF plus ExFms.6His (2.5 ⁇ g/ml).
- the filter inserts with HUVECs were then placed in each well of a 24 well culture plate(lower chamber) containing 600 ⁇ l of DMEM/BSA alone as control, DMEM/BSA plus human recombinant VEGF(10ng/ml), DMEM/BSA/VEGF plus either ExFlk.6His or ExFms.6His and incubated at room temperature for 30 min.
- the migrated cells on the lower surface ofthe filter insert were counted after 4 hours incubation at 37°C.
- Single asterisk indicates significant difference from control (p ⁇ 0.0005) and double asterisk indicates significant difference from VEGF-stimulated (p ⁇ 0.0005).
- the experiment was performed in triplicate and repeated once with similar results. Error bars indicate standard error of all samples.
- FIGS 10A and 10B ExFlk.6His formed heterodimers with endogenous, cell surface Flk-1 in the presence of VEGF.
- ExFlk.6His could function as a "dominant negative" inhibitor, 125j_rf x F V ]
- ⁇ 6His was pre-incubated with either buffer, or an equimolar ratio of VEGF, VEGF plus excess ExFlk.6His, or FGF, and then allowed to bind to the cell surface of cultured endothelial cells(ECRF24).
- Bound ⁇ - ⁇ 1-E Flk .6His was measured by counting lysed cells using a g-counter (Fig. 10A).
- a chemical cross-linker BS3 was added prior to cell lysis. After cross-linking, cell lysates were analyzed on 5%> SDS-PAGE followed by autoradiography (Fig. 10B).
- ExFlk.6His Mechanisms by which ExFlk.6His might inhibit VEGF receptor activation. Endogenous VEGF receptors are activated by ligand- mediated dimerization. Excess ExFlk.6His could function as competitive inhibitor, preventing receptor activation by simply competing for VEGF binding. Alternatively, ExFlk. ⁇ His could also undergo VEGF-mediated heterodimerization with the endogenous receptors on the cell surface and function as a "dominant- negative" inhibitor. It is likely that both mechanisms contribute to the inhibitory action of ExFlk.6His on VEGF stimulated angiogenesis.
- the present invention relates to a soluble endothelium specific receptor inhibitor.
- the inhibitor is a fusion protein comprising an extracellular domain of an endothelium specific receptor fused to a non endothelium specific receptor sequence.
- the present invention further relates to a method of inhibiting angiogenesis using such a fusion protein.
- Endothelium specific receptors of particular relevance to the present invention include those that play a role in endothelial cell proliferation, migration and that are otherwise involved in vascular growth. Examples include Tie-1 (Partanen et al, Mol. Cell. Biol.
- the fusion protein ofthe invention generally comprises an extracellular domain of an endothelium specific receptor, or ligand-binding portion thereof (see, for example, Davis-Smyth, EMBO J. 15:4919 (1996)), advantageously, Tie-2, and a non-endothelium specific receptor sequence.
- the extracellular domain is, preferably, essentially free of transmembrane domain sequences ofthe endothelium specific receptor.
- the non-endothelium specific receptor sequence ofthe fusion protein does not mediate formation of dimers or oligomers and/or is of low immunogenicity (eg is biologically inert, eg compared to Fc).
- the non endothelium specific receptor sequence is, preferably, not Fc.
- Preferred non endothelium specific receptor sequences include polyhistidine (eg a 6-histidine tag) and a strep tag.
- the non receptor sequence is, advantageously, C-terminal to the receptor sequence.
- the present invention also relates to a nucleic acid (DNA or RNA) that encodes the above-described fusion protein.
- the present invention relates to a nucleic acid that encodes a fusion protein comprising the extracellular domain of Tie-2 or KDR (advantageously, human Tie-2 or KDR) fused N-terminal to a 6-His tag.
- the present invention also relates to a recombinant molecule comprising the nucleic acid described above and to a host cell transformed therewith.
- a recombinant molecule comprising a vector and a nucleic acid encoding the fusion protein of the invention can be constructed.
- Vectors suitable for use in the present invention include plasmid and viral vectors.
- Vectors into which a nucleic acid encoding the fusion protein can be cloned include any vector compatible with introduction into a selected host cell.
- Such vectors include any of a wide variety of commercially available plasmids, as well as baculoviruses, retroviruses, and adenoviruses.
- the nucleotide sequence ofthe invention can be present in the vector operably linked to regulatory elements, for example, a promoter. Suitable promoters include strong promoters, including inducible promoters. Specific examples include CMV, PGK and tumor cell specific and/or endothelial cell specific promoters such as Tie-1 or Tie-2 promoters.
- the recombinant molecule ofthe invention can be constructed so as to be suitable for introduction a host cell.
- Suitable host cells include prokaryotic cells, such as bacteria, lower eukaryotic cells, such as yeast, and higher eukaryotic cells, such as mammalian cells, and insect cells.
- the recombinant molecule ofthe invention can be introduced into appropriate host cells by one skilled in the art using a variety of known methods.
- the present invention further relates to a method of producing the fusion protein as defined above.
- the method comprises culturing the above-described host cells under conditions such that the fusion protein encoding sequence is expressed and the fusion protein thereby produced.
- the fusion proteins ofthe invention can be used as antigens to generate fusion protein specific antibodies, which are also within the scope ofthe invention. Methods of antibody generation are well known in the art. Both monoclonal and polyclonal antibodies are included, as are binding fragments thereof.
- the present invention also relates to methods of using the fusion proteins ofthe invention to screen compounds for their ability to bind to the extracellular domain of an endothelium specific receptor and thus to identify compounds that can serve, for example, as agonists or antagonists of angiogenesis.
- a fusion protein ofthe invention comprising a 6-His tag and bound via the tag to a solid support (eg to an affinity column) is contacted with a test compound and the ability ofthe compound to bind the fusion protein determined.
- Test compounds that bind are potential agonists or antagonists of angiogenesis.
- the contacting can be effected in the presence or absence of a natural ligand for the receptor the extracellular domain of which is present in the fusion protein.
- competitive binding assay conditions can be used to determine whether the test compound enhances or inhibits binding ofthe ligand to the fusion protein.
- Compounds that inhibit binding ofthe natural ligand to the fusion protein can be expected to be antagonists of angiogenesis and the reverse for compounds that enhance binding.
- Screening procedures such as those described above are useful for identifying agents for their potential use in the treatment of various forms of pathologic angiogenesis, such as cancer, retinal neovascularization, arthritis and atherosclerosis as well as conditions characterized by reduced vascular density such as diabetes and hypertension.
- pathologic angiogenesis such as cancer, retinal neovascularization, arthritis and atherosclerosis
- Agonists identified in accordance with the above screen can also be used to enhance wound healing.
- the present invention also relates to pharmaceutical compositions comprising, as active agent, the fusion proteins (and nucleic acids) ofthe invention.
- compositions comprising, as active agent, compounds selected using the above-described screening protocols.
- Such compositions include the active agent in combination with a pharmaceutically acceptable carrier.
- the amount of active agent in the composition can vary with the agent, the patient and the effect sought. Likewise, the dosing regimen will vary depending on the composition and the disease/disorder to be treated.
- the present invention also relates to methods of treating pathologic angiogenesis by modulating (e.g., inhibiting) binding of endothelial growth factors to endothelium specific receptors so as to preclude receptor activation (eg dimerization).
- This embodiment ofthe invention includes the use in gene therapy regimens of DNA sequences encoding the above-described fusion proteins.
- the encoding sequences can be present in a construct which, when introduced into target cells, results in expression ofthe DNA sequence and production ofthe fusion protein.
- Target cells include endothelial cells, particularly endothelial cells present at the site of pathologic angiogenesis (eg, at a tumor site, retina, synovium, atherosclerotic plaque.)
- target cells can be distant from the site of pathologic angiogenesis so long as expression ofthe DNA results in circulating levels ofthe fusion protein sufficiently high to exert the effect sought (that is, enhancement or inhibition of angiogenesis).
- a DNA transfer method that: (1) directs the therapeutic sequence into specific target cell types, (2) is highly efficient in mediating uptake ofthe therapeutic polynucleotide into the target cell population, and (3) is suited for use in vivo for therapeutic application.
- Delivery ofthe fusion protein encoding sequence can be effected using any of a variety of methodologies including transfection with a viral vector, fusion with a lipid, and cationic supported DNA introduction (see generally Verma et al, Nature 389:239 (1997)).
- transfection with a viral vector fusion with a lipid
- cationic supported DNA introduction see generally Verma et al, Nature 389:239 (1997).
- Adenoviral vectors have been described for use in human gene therapy. Advantages of adenovirus vectors, particularly replication defective adenoviruses, include safety, the potential to carry large insert polynucleotide sequences, very high viral titres, ability to infect non-replicating cells, and suitability for infecting tissues in situ.
- adenoassociated viruses which integrate, can be used, as can other viral systems depending on the target site, or natural/engineered tissue tropism.
- Gene transfer can be effected using replication-defective retroviral vectors harboring the therapeutic polynucleotide sequence as part ofthe retroviral genome (Miller et al, Mol. Cel. Biol. 10:4239 (1990); Kolberg, J. NIH Res. 4:43 (1992); Cornetta et al, Hum. Gene. Ther. 2:215 (1991)).
- Advantages of retroviral vectors for gene therapy include the high efficiency of gene transfer into replicating cells, the precise integration ofthe transferred genes into cellular DNA, and the lack of further spread ofthe sequences after gene transduction.
- plasmid DNA can be purified to homogeneity thereby reducing the potential for pathogenic contamination.
- Liposome-mediated DNA transfer has been described by various investigators (Wang and Huang, Biochem. Biphys. Res. Commun. 147:980 (1987); Wang and Huang, Biochemistry 28:9508 (1989); Litzinger and Huang, Biochem. Biophys. Acta 1113:201 (1992); Gao and Huang, Biochem. Biophys. Res. Commun. 179:280 (1991); Feigner, WO 91/17424; WO 91/16024).
- Liposomal compositions may not possess the specificity necessary to deliver the exogenous DNA to all target cell types and non-physiological pH conditions may be necessary to effect fusion.
- Immunoliposomes have also been described as carriers of exogenous polynucleotides (Wang and Huang, Proc. Natl. Acad. Sci. USA 84:7851 (1987); Trubetskoy et al, Biochem. Biophys. Acta 1131 :311 (1992)). Immunoliposomes can be expected to have improved cell type specificity as compared to liposomes due to the inclusion of specific antibodies that bind to surface antigens on target cell types.
- Low molecular weight polylysine and other polycations are carriers that can be used to effect DNA-mediated transfection into cells.
- Zhou et al (Biochem. Biophys. Acta 1065:8 (1991)) have reported synthesis of a polylysine - phospholipid conjugate, a lipopolylysine comprising PL linked to N-glutarylphosphatidylethanolamine, which reportedly increases the transfection efficiency of DNA as compared to lipofectin, a commercially used transfection reagent.
- any suitable DNA delivery method can be used in the context ofthe present invention, including direct physical application of naked DNA comprising the expression construct/transgene to the target cell population.
- the nucleic acid-containing compositions ofthe invention can be stored and administered in a sterile physiologically acceptable carrier, where the nucleic acid is dispersed in conjunction with any agents which aid in the introduction of the DNA into cells.
- a sterile physiologically acceptable carrier including water, PBS, ethanol, lipids, etc.
- concentration ofthe DNA will be sufficient to provide a therapeutic dose, which will depend on the efficiency of transport into the cells.
- compositions containing the present fusion protein encoding sequences can be administered for prophylactic and/or therapeutic treatments.
- compositions are administered to a patient already affected by the particular disease/disorder, in an amount sufficient to cure or at least partially arrest the condition and its complications.
- An amount adequate to accomplish this is defined as a "therapeutically effective dose” or "efficacious 98/18914
- Amounts effective for this use will depend upon the severity ofthe condition, the general state ofthe patient, and the route of administration. It will be appreciated that combinations of fusion protein encoding sequences ofthe invention can be used, for example, sequences encoding the ExTek.6His and ExFlk.6His fusion proteins described in the Examples that follow.
- the fusion protein ofthe invention can be used directly to inhibit angiogenesis.
- the fusion protein can be administered, for example, systemically or locally, by injection, infusion, via a slow release device, or via a pump. While the amount administered and the dosing regimen will vary with the protein, the mode of administration, the patient and the effect sought, cancer treatment may be effected using for example, 50 ⁇ g-2mg doses, administered by IV infusion over 30 min to 1 hr, twice weekly, similarly arthritis treatment. Arthritis treatment can also be effected via local administration to the involved synovium.
- the fusion protein can be administered intraocularly via injection (eg at doses of 100 ng to 100 ⁇ g), scleral pumps can also be used.
- Atherosclerosis can be treated with systemic administration ofthe fusion protein or administration via a stent or balloon angioplasty.
- the invention includes the administration of more than one fusion protein ofthe invention.
- the rat endothelial marker MRC-OX43 and biotinylated anti-mouse immunoglobulin was purchased from Harlan Bioproducts for Science and Dako Corporation, respectively.
- HRP conjugated Streptavidin and liquid DAB substrate kits were from Biogenex Corporation.
- Avidin Biotin blocking and DAB enhancing solution were from Vector.
- mice Tek-specific cDNA primers one : 5' GGATCCATGGACCTGATC 3', an Bam H I site was introduced in front ofthe start codon; the other: 3* CGTCTGGAGCCTAGCTA 5', a Cla I site was introduced at 5' end
- the cDNA from nucleotide 124 to 2346 or amino acids 1- 741 of mouse Tie2 was amplified by RT-PCR from 9-12 day mouse embryo as previously described (Dumont et al, Oncogene 8:1293-1301 (1993)).
- BSK Cla-/6His is a modified BSK vector (Stratagene). The original Cla I site was deleted and a new Cla I site with an in frame 6-histidine tag followed by a stop codon was inserted into the BamH I and Xba I site. A 2.1 kb EcoR I/Not I fragment from
- BSK ExTek.6His containing the mouse extracellular domain coding region followed by a six histidine tag was subcloned into the same sites of pVL 1393, a baculoviral expression vector (Pharmingen).
- the ExTek.6His transfer plasmid and Baculogold baculoviral DNA (Pharmingen) were co-transfected into SF9 cells for production ofthe recombinant baculovirus BvExTek. ⁇ His according to the manufacturer's instructions.
- Second passage virus was used to infect serum- free SF9 cells for ExTek.6His protein expression.
- the same approach was used to generate a recombinant baculovirus (BvExFms. ⁇ His) expressing the entire extracellular domain of human c-fms receptor fused to a 6-histidine tag at C terminus.
- Mock control solution was generated from the supernatant of uninfected SF9 cells following the same purification procedure as for ExTek.6His protein. Aliquots of purified ExTek.6His and ExFms.6His protein and mock control solution were analyzed by SDS-PAGE on a 7.5% gel.
- Tumor window chamber model Tumors were grown in cutaneous window chambers in Fischer 344 rats
- a 0.1 mm3 piece of R3230AC tumor from a donor rat was then placed onto the fascial plane and an additional 100 ⁇ l of ExTek.6His protein or control solution was added and the chambers were sealed with glass cover slips.
- the tissue within the chamber is approximately 200 microns thick and is semi-transparent.
- a pair of tumor window chambers were done at each time, one treated with ExTek.6His and the other with mock control solution.
- the tumor implants in each pair were tailored in similar size from the same larger piece of grossly viable tumor tissue.
- the areas of tumor implants were measured by using an image analysis software (JAVA, Jandel Co.). The baseline host vasculature, the blood flow and the proximity of tumor tissue to vessels were scored.
- Tumor growth and neovascularization was photographed using a dissecting microscope (Zeiss, Stemi SV6) on days 5, 7 and 10 and window chambers were harvested for H & E staining on day 10.
- a window chamber bearing a 5 day old untreated R3230AC tumor was freshly frozen in OCT.
- Tumor volumes which were assumed to approximate a flat cylinder in shape, were calculated using the formula:
- Tumor vascular length density as an indicator of tumor vasculature was measured from photographs of 10 day old tumors within the window chamber using a previously described method (Dewhirst et al, Radiat. Res. 130:345-354 (1992)). 3-5 areas inside the tumor were randomly selected for measurement. The vascular length density in mm/rrnrP was calculated using the formula:
- pellets were rehydrated with a drop of PBS buffer and then placed in a surgically created pocket within the cornea stroma, 1.5 mm from the limbus. Corneas were observed every other day until day 5 or 7 when the animals were anesthetized and perfused with lactated ringers solution followed by colloid carbon solution to enumerate the vessels. Responses were scored as positive when vigorous and sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant were detected. Negative responses were recorded when no growth was detected or when there was only an occasional sprout or hairpin loop with no evidence of sustained growth. Positive controls consisted of Hydron pellets containing 25 ng/5 ⁇ l pellet.
- Negative controls consisted of sham implants and Hydron pellets containing media alone. Media was incorporated into pellets at a concentration of 1 ⁇ g of total protein per cornea. Representative corneas were examined histologically and except for occasional neutrophils found in the limbus of both control and test corneas, nonspecific inflammation was not a contributing factor in any ofthe corneal responses.
- Tumor windows to be processed for H & E staining were fixed in 4%> paraformaldehyde overnight at 4°C and embedded in paraffin.
- Samples for immunohistochemistry were freshly frozen in liquid nitrogen and embedded in OCT.
- serial sections of 10 microns were cut, fixed in ice cold acetone for 10 minutes, and blocked with 0.03 %> H2O2, 1% horse serum and avidin/biotin blocking reagents (Vector). After blocking, sections were incubated with a monoclonal anti-Tie2 antibody or an antibody specific for rat endothelium (MRC-OX43) in a humidified chamber for 50 minutes at room temperature.
- MRC-OX43 monoclonal anti-Tie2 antibody or an antibody specific for rat endothelium
- ExTek.6His and ExFms.6His were purified from the supernatant of baculovirus infected SF9 cells by one-step
- ExTek.6His blocks angiogenesis stimulated by tumor cell conditioned media
- n m (x):m is the number of rats used in each experiment; n is the number of rats with the indicated response; x is the percentage of rats with the indicated response.
- Tie2 is expressed in tumor vessels at the onset of tumor angiogenesis
- ExTek. ⁇ His protein was used as an inhibitor in a rat cutaneous window chamber bearing a R3230AC mammary tumor.
- small fragments of tumor O.lmm ⁇
- vascularization of tumors in the window chamber is first detected at about 5 days after implantation and is followed by a rapid growth phase ofthe tumor.
- immunohistochemical studies demonstrated expression of Tie2 in vessels surrounding and penetrating the tumor implant at 5 days after implantation.
- ExTek inhibits tumor growth in cutaneous "window chambers"
- Recombinant adenovirus was generated and propagated in monolayer cultured 293 cells maintained in DMEM supplemented with 10%> fetal bovine serum and 1% penicillin/streptomycin (Gibco/BRL) at 37°C with 5% C ⁇ 2-
- the murine mammary carcinoma cell line 4T1 and murine melanoma cell line F10.9 were maintained in DMEM plus 10%> fetal bovine serum at 37°C with 5% CO2.
- a novel adeno viral vector system in which partial El region and all the E3 region were deleted, derived from the in340 strain of adenovirus type 5 (ad5) was used in construction of AdPac ⁇ -gal (Channon et al, Cardiovascular Res. 32:962-972 (1996)). The ⁇ -gal gene was inserted in El region. The resulting viral vector, AdPac ⁇ -gal, served as a control virus and was used to generate AdExTek by replacing the ⁇ -galactosidase gene with one containing ExTek cDNA.
- the ExTek cDNA containing the mouse extracellular domain coding region fused to a strep-tag at C terminal was subcloned into BamH I and Nhe I site of the transfer plasmid pGEM CMV/BGH poly(A).
- the plasmid pGEM CMV/BGH poly(A).
- CMV ExTek/BGH poly (A) was then digested with Xba I and Bst B 1 , and the 3.6 kb plasmid fragment consistent with the early CMV promotor and enhancer, ExTek and BGH was directly ligated to the Xba I site of parent virus Ad.Pac ⁇ -gal DNA.
- the ligation mixture was purified by phenol-chloroform extraction and ethanol precipitation, then transfected into 293 cells using the calcium phosphate method. After observation of a cytopathathic effect (7-10 days), the cells were lysed by multiple freeze/thaw cycles, and the recombinant virus was isolated by plaque assay on 293 cells.
- Plaque purified AdExTek or Ad.Pac ⁇ -gal virus was used to generate high titer virus stock by infecting forty 150 mm plates of confluent 293 cells at a multiplicity of infection of 2 in DMEM plus 2%o FBS.
- the viruses were purified from the infected cell lysates as described previously (Channon et al, Cardiovascular Res. 32:962-972 (1996)).
- the virus was stored in virus storage buffer (VSB; 20 mM Tris pH 7.4, 150 mM NaCl, 5 mM KCL, 1 mM MgCl 2 ) plus
- ExTek concentration in blood was determined by a simple ELISA assay.
- AdExTek adenovirus (5x10° pfu) was injected into Balb/c mouse circulation through retro-obital sinus. Two days later, small amount of blood was collected in a heparinized microcapiUary tube from tail vein. Mouse plasma was recovered after brief centrifugation to remove cells. Serial dilutions (in PBS) of the plasma were incubated in micro wells overnight at 40°C. The coated wells were blocked with 5 % milk in TBST (lOmM of Tris Cl, pH 8.0, 150mM of NaCl, 0.05% of Tween 20) for 30 minute.
- a biotinylated mouse anti Tie2 monoclonal antibody (Ab33) diluted in TBST (0.5 ⁇ g/ml) was incubated for 1 hour followed by incubation with 1 :2000 diluted strep-avidin alkaline phosphatase conjugate (Gibco/BRL) in TBST for 30 minutes.
- the phosphatase activity was determined by addition of Sigma @104 phosphatase substrate dissolved in diethanolamine and 0.5 mM MgCl2, pH 9.8, and absorbency at 405nm was measured in V max Kinetic Microplate Reader
- a murine mammary tumor cell line 4T1 or a murine melanoma cell line F10.9 was implanted into the flank of female Balb/c mouse or
- mice lungs to be processed for H & E staining were fixed in Bouin's solution for a few days, decolorized in 70%) ethanol several times before being embedded in paraffin. Serial sections of 8 microns were cut. The sections were stained with H&E for histoligic examination.
- AdExTek a recombinant adenovirus for gene transfer of ExTek
- AdExTek a recombinant adenovirus
- ExTek cDNA was inserted in El region driven by the CMV promotor.
- AdExTek was administered to Balb/c mice in intravenous injection into the retro- orbital sinus. The plasma was collected every other day after viral injection. Serial dilutions ofthe plasma were then incubated in ELISA plates. The ExTek content was determined using biotinylated anti-Tie2 antibody (Ab33). The concentration of ExTek was calibrated using purified recombinant ExTek (Lin et al, J. Clin. Invest. 100:2072-2078 (1997)). High level expression could be detected in the plasma (1-2 mg/ml) two days after viral injection. The high level expression was transient with levels falling to baseline within 12 days (Fig 5B). The mice with high levels of circulating ExTek showed no apparent ill effects, no weight loss and no behavioral abnormalities. AdExTek inhibits the growth rate of two well established primary murine tumors
- Tumor cells 5x10 ⁇ in 50 ⁇ l of PBS were implanted into the flank of female Balb/c mice (for 4T1) or female C57/BL (for F10.9). Following the development of an easily palpable tumor (7-
- AdExTek suppress tumor metastasis
- two highly metastatic cell lines 4T1 and F10.9 (5x10 ⁇ ) were mixed with 5x10 ⁇ pfu of either the AdExTek virus or the control AdPac ⁇ -gal virus and then co-injected into the circulation through retro-obital sinus.
- 5x10 ⁇ 5x10 ⁇
- 3 mice injected with control virus died from massive lung metastases but all ofthe mice that received AdExTek were alive.
- the remaining animals were sacrificed and the lungs were removed, weighed and fixed in Bouin's solution. Lungs were grossly examined under dissecting microscopy.
- Recombinant baculovirus was generated and propagated in monolayer cultured SF9 cells maintained in Grace's Insect Medium Supplemented (Gibco/BRL) at 28°C. Protein expression was carried out in suspension cultured SF9 cells in Protein-Free Insect Medium (Gibco/BRL).
- the R3230AC rat adenocarcinoma cell line was maintained in DMEM plus 10%> fetal bovine serum (FBS, Gibco/BRL) at 37°C with 5% CO2- Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics Inc.
- ECRF24 an immortalized HUVEC, was provided by Dr. Hans Pannekoek (Fontijn et al, DNA Exp. Cell.
- Endothelial cells were maintained at 37°C, 5% CO 2 in complete endothelial cell growth medium (EGM, Clonetics, Inc) and grown on 2% gelatin (Sigma) coated plates. Endothelial cells were serum starved in endothelial cell basal medium (EBM, Clonetics, Inc). Anti-Flk antibodies (C-20 and 1158) and protein-A agarose were purchased from Santa Cruz Biotechnology. Anti phosphotyrosine antibody (4G10) was purchased from Upstate Biotechnology. Human recombinant VEGF 165 was purchased from R & D Systems. Construction ofthe ExFlk.6His and ExFms.6His baculovirus vectors and production of recombinant viruses
- VEGF vascular endothelial growth factor
- flk-1 and flt-1 Two closely related endothelium-specific receptor tyrosine kinases, flk-1 and flt-1 (De Vries et al, Science 255:989-991 (1992), Terman et al, Biochem. Biophys. Res. Comm. 187:1579-1586 (1992), Shibuya, Adv. Cancer Res. 67:281-316 (1995)).
- the entire extracellular domain of murine flk-1 was generated by PCR from a plasmid containing a cDNA encoding the extracellular and transmembrane domains of flk-1.
- the forward primer consisted of nucleotides 208 to 223 with a BamHI site introduced upstream ofthe start codon; the reverse primer consisted of nucleotides 2493-2478 in the reverse orientation with a Cla I site introduced at the 5' end.
- the resulting PCR product was digested with BamHI/Clal and ligated to the same sites of an intermediate vector (BSK Cla-/6His) to generate a cDNA encoding a fusion protein consisting ofthe entire extracellular domain of flk-1 with a 6 histidine tag at the C terminus
- BSK/ExFlk.6His A 2.3 kb EcoR I/Not I fragment from BSK/ExFlk.6His was subcloned into the same sites of pVL 1393, a baculoviral expression transfer vector (Pharmingen).
- pVL 1393/ExFlk.6His and Baculogold baculoviral DNA were co-transfected into SF9 cells for production ofthe recombinant baculovirus (BvExFlk.6His) according to the manufacturer's instructions. Second passage virus was used to infect serum-free SF9 cells for ExFlk.6His protein production.
- Suspension cultured SF9 serum free insect cells (1 liter) were infected with approximately lpfu cell of second passage BvExFlk.6His or BvExFms.6His for 54 hours at 28°C. Cells were removed by centrifugation at 3000 rpm(Sorvall) for 20 minutes at 4°C. The supernatant was dialyzed against 8 liters of PBS pH 8.0 for 48 hours with one change of buffer. The dialyzed supernatant was then incubated with 4ml of Ni ++ NTA resin (Qiagen). After 1 hour at room temperature, the resin-bound ExFlk.6His protein or ExFms.6His protein was loaded onto a 10ml column.
- wash buffer 50mM NaH2PO. ⁇ , 300mM NaCl and 20mM imidazole pH 8.0
- the protein was eluted with elution buffer (wash buffer plus 250mM imidazole) followed by a buffer change to PBS pH 7.2 by ultra filtration (Centricon 10 from Amicon Co.).
- Mock control material used in the tumor study was generated from the supernatant of uninfected SF9 cells following the same purification procedure. Aliquots of purified ExFlk.6His and ExFms.6His proteins were analyzed by SDS- PAGE on a 7.5% gel.
- the wells were washed once with blocking buffer (25mM Hepes pH 7.4, lOOmM NaCl, 0.5% gelatin, 20 ⁇ g/ml BSA) and then blocked with the same buffer for 2 hours at room temperature.
- Increasing amounts of 125j labeled ExFlk. ⁇ His (0.03-300nM) in binding buffer (25mM Hepes pH7,4, lOOmM NaCl, 20 ⁇ g/ml BSA, 0.5 ⁇ g/ml heparin) were added to each well and incubated for 1 hour at room temperature.
- Nonspecific binding was determined by incubation of 125 I-labeled ExFlk. ⁇ His in the presence of 60-fold excess of unlabeled ExFlk. ⁇ His.
- the wells were washed 3 times with binding buffer and counted in a gamma counter. Scatchard analysis of binding was performed with the aid ofthe Scatchard Fit program.
- ECRF cells a transformed HUVEC line expressing Flk-1, were grown in EGM in a 60 mm dish (Fontijn et al, DNA Exp. Cell. Res. 216:199-207 (1995)). Upon reaching confluence, the cells were serum starved in EBM overnight followed by stimulation with human recombinant VEGF at 20 ng/ml plus different amounts of ExFlk. ⁇ His at 37°C for 5 minutes.
- the cells were then washed with ice cold PBS 3 times on ice and then lysed with lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1 % triton X- 100, 2 mM EDTA) supplemented with 1 mM PMSF (a protease inhibitor), 10 ng/ml leupeptin, 1 mM sodium vanadate.
- Flk was immunopreciptated with anti-Flk antibody (antibody 1158, Santa Cruz) for 4 hours at 4°C, and immuncomplexes were collected by addition of protein A agarose beads.
- HUVECs were plated in 24 well plates at a density of 25000 cells per well. After 24 hours, the media was replaced by endothelial cell basal media (EBM as quiescence media, Clonetics, Inc) and incubated for an additional 24 hours. The old media was then replaced with either fresh quiescence media alone as a control, or quiescence media plus human recombinant VEGF at 1 Ong/ml, or quiescence media plus 1 Ong/ml VEGF and purified ExFlk. ⁇ His at 2.5 ⁇ g/ml, or quiescence medium plus lOng/ml VEGF and purified control ExFms. ⁇ His at 2.5 ⁇ g/ml.
- the cells were incubated for 24 hours followed by a 3 hour pulse-labeling with 2 ⁇ Ci/ml 3 H-thymidine (Amersham). The reaction was stopped by aspirating the media and washing the cells with Hanks Balanced Salt Solution (Gibco/BRL). The DNA was precipitated by treating the cells with cold 10% TCA at 4°C for 30 min followed by an absolute ethanol wash. The precipitated material was resuspended in 0.5ml of 0.5M NaOH, and ⁇ H-thymidine incorporation was determined from a 400 ⁇ l aliquot using Beckman LS6000SC scintillation counter.
- the rate of migration of HUVECs was determined by using a modified Boyden chamber assay as described by Clyman et al (Cell Adhesion and Commun. 1 :333 (1994)). Briefly, Polycarbonate filter wells (Costar Transwell with an 8 ⁇ m pore size) were coated with 2% gelatin in PBS for 30 min at room temperature and subsequently incubated at 37°C for 1 hour with DMEM containing 0.1% BSA (DMEM/BSA). Confluent HUVECs were trypsinized, pelleted by centrifugation, washed with DMEM/BSA to remove residual serum and resuspended in fresh DMEM BSA to a final concentration of 2 x 10 6 cells/ml.
- the lysate was recovered after centrifugation to remove cell nuclei and either directly analyzed by 5%> SDS- PAGE followed by auto radiography or immunopreciptated with an anti-Flk antibody raised against a c-tail peptide of Flk (antibody c-20, Santa Cruz) before the SDS-PAGE analysis.
- pellets were rehydrated with a drop of PBS and then placed in a surgically created pocket within the corneal stroma, 1.5mm from the limbus. Corneas were observed every other day until day 5 or 7 when the animals were anesthetized and perfused with lactated ringers solution followed by colloidal carbon solution to enumerate the vessels. Responses were scored as positive when vigorous and sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant were detected. Negative responses were recorded when no growth was detected or when there was only an occasional sprout or hairpin loop with no evidence of sustained growth. Negative controls consisted of Hydron pellets containing media alone. Media was incorporated into pellets at a concentration of 1 ⁇ g of total protein per pellet.
- Soluble receptor proteins were incorporated into pellets at lOOng per pellet. Histological examination of representative corneas revealed that nonspecific inflammation was not a contributing factor in any ofthe corneal responses except for occasional neutrophils found in the limbus of both control and test corneas.
- Tumor window chamber model R3230AC tumors were grown in cutaneous window chambers in Fischer
- a 0.1mm 3 piece of tumor from a donor rat was then placed onto the fascial plane and an additional lOO ⁇ l of protein or control solution was added and the chambers were sealed with glass cover slips.
- a pair of tumor window chambers were done at each time, one treated with ExFlk. ⁇ His and the other with control solution.
- the tumor implants in each pair were taken from the same region of grossly viable donor tumor tissue.
- Ten days after implantation, tumors in window chambers (200 ⁇ m thick) were photographed using transillumination and a dissecting microscope (Zeiss, Stemi SV6) for vascular length density measurement and were subsequently harvested for H & E staining.
- Tumor volumes which were assumed to approximate a flat cylinder in shape, were calculated using the formula:
- Tumor volume (mm3) 3.14t(d/2)2
- Tumor vascular length density as an indicator of tumor vascularity was measured from photographs of 10 day old tumors within the window chamber using a previously described method (Dewhirst et al, Radiat. Res. 130:345-354 (1992)). 3-5 areas inside the tumor were randomly selected for measurement. The vascular length density in mm/mm 3 was calculated using the formula:
- R3230AC tumor cells were seeded at 2X10 4 /well into 24-well plates and maintained in the presence of purified ExFlk. ⁇ His protein (3 ⁇ M) or control solution. Cell morphology was monitored each day by light microscopy. Cells were trypsinized, suspended in PBS containing 0.02% trypan blue (Gibco/BRL) and live cells were counted with a hemacytometer on each following day for three days.
- Results are reported as mean ⁇ SE for tumor volume and tumor vascular length density for each group.
- a two-tailed Student's t test was used to analyze statistical differences between control and ExFlk. ⁇ His treated groups. Differences were considered statistically significant at p ⁇ 0.05.
- ExFms. ⁇ His was purified from the supernatant of SF9 cells infected with a baculovirus vector directing the expression of a fusion between the extracellular domain ofthe CSF-1 receptor, c-fms, fused to a 6 histidine-tag.
- a mock control was purified from uninfected SF9 cells using the same protocol.
- ExFlk. ⁇ His and ExFms. ⁇ His proteins were purified to near homogeneity, yielding a single major band with the expected molecular masses of approximately 105kD and 70kD, respectively (Figure 8B).
- ExFlk.6His protein binds VEGF, blocks the activation of endothelial VEGF receptor and neutralizes VEGF stimulated endothelial proliferation and migration in vitro
- binding assays were done by adding 125j labeled ExFlk. ⁇ His to microtiter wells pre- coated with human recombinant VEGF. Bound 125j ExFlk. ⁇ His was then detected by counting the washed wells in a Gamma counter. Under the conditions 8/18914
- ECRF endothelial cells
- ExFlk. ⁇ His protein (2.5 ⁇ g/ml) but was not blocked by ExFms. ⁇ His at the same concentration (p ⁇ 0.0005).
- HUVEC migration rate in a modified Boyden chamber assay increased approximately 3 fold by the addition of VEGF at 1 Ong/ml (p ⁇ 0.0005), and this increase in migration was reduced to background levels after pre-incubation of VEGF with ExFlk. ⁇ His (2.5 ⁇ g/ml) but not with ExFms. ⁇ His at the same concentration (p ⁇ 0.0005, Fig. 9C).
- ExFlk.6His forms a VEGF-dependent heterodimer with endogenous VEGF receptors on the surface of cultured endothelial cells
- ' ⁇ ⁇ l-ExFlk. ⁇ iis was mixed with VEGF to form a 125j_r ⁇ Fik .6His/VEGF complex. This pre-formed complex was then tested for its ability to bind to VEGF receptors on the surface of cultured endothelial cells (Figure 10A).
- the cross- linked complex was immunopreciptated with an antibody against the c-tail of Flk- 1 (antibody 1158, Santa Cruz) and analyzed by SDS-PAGE and autoradiography. Again, consistent with the formation of VEGF mediated receptor heterodimer, a single high molecular complex containing both the endogenous cell surface receptor and the radiolabeled soluble receptor was immunopreciptated only in the presence of VEGF.
- ExFlk. ⁇ His could function as a "dominant negative" inhibitor of VEGF receptor activation and should be a potent inhibitor of angiogenesis in vivo.
- ExFlk.6His inhibits corneal angiogenesis in vivo
- ExFlk. ⁇ His (lOOng)
- the angiogenic response induced by tumor conditioned media was totally blocked in 5 corneas (72%>) and a weak growth was seen in 2 corneas (28%).
- Addition of a control protein, ExFms. ⁇ His, to pellets containing tumor cell conditioned media did not block vessel formation.
- vascularization of tumors in the window chamber is first detected at about 5 days after implantation and is followed by a rapid growth phase ofthe tumor.
- ExFlk.6His inhibits tumor vascularization
- ExFlk. ⁇ His protein This finding is in accordance with the ability of ExFlk. ⁇ His protein to neutralize VEGF mediated endothelial responses in vitro, and to block tumor cell conditioned media stimulated angiogenesis in the rabbit cornea. It is also consistent with the notion that the primary action ofthe ExFlk. ⁇ His protein is to inhibit tumor neovascularization. ExFlk.6His does not directly affect tumor cell proliferation or viability
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
L'invention concerne un récepteur Tie2 soluble et son utilisation en tant qu'agent antiangiogénique.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU51540/98A AU5154098A (en) | 1996-10-31 | 1997-10-31 | Soluble tie2 receptor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US2940796P | 1996-10-31 | 1996-10-31 | |
US60/029,407 | 1996-10-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998018914A1 true WO1998018914A1 (fr) | 1998-05-07 |
Family
ID=21848847
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/019597 WO1998018914A1 (fr) | 1996-10-31 | 1997-10-31 | Recepteur tie2 soluble |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU5154098A (fr) |
WO (1) | WO1998018914A1 (fr) |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6413932B1 (en) | 1999-06-07 | 2002-07-02 | Immunex Corporation | Tek antagonists comprising soluble tek extracellular binding domain |
US6521424B2 (en) | 1999-06-07 | 2003-02-18 | Immunex Corporation | Recombinant expression of Tek antagonists |
WO2006002854A3 (fr) * | 2004-06-25 | 2006-03-16 | Licentia Ltd | Recepteurs tie et ligands pour le tie, et procedes de modulation de la fertilite feminine |
US7521053B2 (en) | 2001-10-11 | 2009-04-21 | Amgen Inc. | Angiopoietin-2 specific binding agents |
US7658924B2 (en) | 2001-10-11 | 2010-02-09 | Amgen Inc. | Angiopoietin-2 specific binding agents |
US8071650B2 (en) | 2000-08-21 | 2011-12-06 | Pacific Corporation | Thiourea derivatives and the pharmaceutical compositions containing the same |
WO2012162561A2 (fr) | 2011-05-24 | 2012-11-29 | Zyngenia, Inc. | Complexes plurispécifiques multivalents et monovalents, et leurs utilisations |
EP2671891A2 (fr) | 2008-06-27 | 2013-12-11 | Amgen Inc. | Inhibition d'ang-2 pour traiter la sclérose en plaques |
US9795594B2 (en) | 2006-06-27 | 2017-10-24 | Aerpio Therapeutics, Inc. | Human protein tyrosine phosphatase inhibitors and methods of use |
US9926367B2 (en) | 2006-04-07 | 2018-03-27 | Aerpio Therapeutics, Inc. | Antibodies that bind human protein tyrosine phosphatase beta (HPTPbeta) and uses thereof |
US9949956B2 (en) | 2009-07-06 | 2018-04-24 | Aerpio Therapeutics, Inc. | Compounds, compositions, and methods for preventing metastasis of cancer cells |
US9994560B2 (en) | 2014-03-14 | 2018-06-12 | Aerpio Therapeutics, Inc. | HPTP-β inhibitors |
US10150811B2 (en) | 2011-10-13 | 2018-12-11 | Aerpio Therapeutics, Inc. | Methods for treating vascular leak syndrome and cancer |
EP3424530A1 (fr) | 2013-03-15 | 2019-01-09 | Zyngenia, Inc. | Complexes multispécifiques monovalents et multivalents et leurs utilisations |
US10220048B2 (en) | 2013-03-15 | 2019-03-05 | Aerpio Therapeutics, Inc. | Compositions and methods for treating ocular diseases |
US10336820B2 (en) | 2008-02-20 | 2019-07-02 | Amgen Inc. | Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof |
US10952992B2 (en) | 2015-09-23 | 2021-03-23 | Aerpio Pharmaceuticals, Inc. | Methods of treating intraocular pressure with activators of Tie-2 |
US11253502B2 (en) | 2019-04-29 | 2022-02-22 | EyePoint Pharmaceuticals, Inc. | Tie-2 activators targeting the Schlemm's canal |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5338669A (en) * | 1989-09-20 | 1994-08-16 | Abbott Biotech, Incorporated | Method of producing fusion proteins |
US5405941A (en) * | 1993-04-15 | 1995-04-11 | National Jewish Center For Immunology And Respiratory Medicine | MEKK protein, capable of phosphorylating MEK |
US5447860A (en) * | 1992-06-26 | 1995-09-05 | Immunex Corporation | Tyrosine kinase |
US5488032A (en) * | 1987-11-25 | 1996-01-30 | Immunex Corporation | Method of using soluble human interleukin-1 receptors to suppress inflammation |
-
1997
- 1997-10-31 WO PCT/US1997/019597 patent/WO1998018914A1/fr active Application Filing
- 1997-10-31 AU AU51540/98A patent/AU5154098A/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5488032A (en) * | 1987-11-25 | 1996-01-30 | Immunex Corporation | Method of using soluble human interleukin-1 receptors to suppress inflammation |
US5338669A (en) * | 1989-09-20 | 1994-08-16 | Abbott Biotech, Incorporated | Method of producing fusion proteins |
US5447860A (en) * | 1992-06-26 | 1995-09-05 | Immunex Corporation | Tyrosine kinase |
US5405941A (en) * | 1993-04-15 | 1995-04-11 | National Jewish Center For Immunology And Respiratory Medicine | MEKK protein, capable of phosphorylating MEK |
Non-Patent Citations (1)
Title |
---|
MOLECULAR AND CELLULAR BIOLOGY, April 1992, Vol. 12, No. 4, PARTANEN et al., "A Novel Endothelial Cell Surface Receptor Tyrosine Kinase with Extracellular Epidermal Growth Factor Homology Domains", pages 1698-1707. * |
Cited By (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003501090A (ja) * | 1999-06-07 | 2003-01-14 | イミュネックス・コーポレーション | Tekアンタゴニスト |
US6521424B2 (en) | 1999-06-07 | 2003-02-18 | Immunex Corporation | Recombinant expression of Tek antagonists |
US7067475B2 (en) | 1999-06-07 | 2006-06-27 | Immunex Corporation | Tek antagonists |
US6413932B1 (en) | 1999-06-07 | 2002-07-02 | Immunex Corporation | Tek antagonists comprising soluble tek extracellular binding domain |
US8071650B2 (en) | 2000-08-21 | 2011-12-06 | Pacific Corporation | Thiourea derivatives and the pharmaceutical compositions containing the same |
US7521053B2 (en) | 2001-10-11 | 2009-04-21 | Amgen Inc. | Angiopoietin-2 specific binding agents |
US7658924B2 (en) | 2001-10-11 | 2010-02-09 | Amgen Inc. | Angiopoietin-2 specific binding agents |
WO2006002854A3 (fr) * | 2004-06-25 | 2006-03-16 | Licentia Ltd | Recepteurs tie et ligands pour le tie, et procedes de modulation de la fertilite feminine |
US11814425B2 (en) | 2006-04-07 | 2023-11-14 | Eye Point Pharmaceuticals, Inc. | Antibodies that bind human protein tyrosine phosphatase beta (HPTPbeta) and uses thereof |
US9926367B2 (en) | 2006-04-07 | 2018-03-27 | Aerpio Therapeutics, Inc. | Antibodies that bind human protein tyrosine phosphatase beta (HPTPbeta) and uses thereof |
US9795594B2 (en) | 2006-06-27 | 2017-10-24 | Aerpio Therapeutics, Inc. | Human protein tyrosine phosphatase inhibitors and methods of use |
USRE46592E1 (en) | 2006-06-27 | 2017-10-31 | Aerpio Therapeutics, Inc. | Human protein tyrosine phosphatase inhibitors and methods of use |
US10463650B2 (en) | 2006-06-27 | 2019-11-05 | Aerpio Pharmaceuticals, Inc. | Human protein tyrosine phosphatase inhibitors and methods of use |
US10336820B2 (en) | 2008-02-20 | 2019-07-02 | Amgen Inc. | Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof |
EP2671891A2 (fr) | 2008-06-27 | 2013-12-11 | Amgen Inc. | Inhibition d'ang-2 pour traiter la sclérose en plaques |
US9949956B2 (en) | 2009-07-06 | 2018-04-24 | Aerpio Therapeutics, Inc. | Compounds, compositions, and methods for preventing metastasis of cancer cells |
WO2012162561A2 (fr) | 2011-05-24 | 2012-11-29 | Zyngenia, Inc. | Complexes plurispécifiques multivalents et monovalents, et leurs utilisations |
US10150811B2 (en) | 2011-10-13 | 2018-12-11 | Aerpio Therapeutics, Inc. | Methods for treating vascular leak syndrome and cancer |
US10815300B2 (en) | 2011-10-13 | 2020-10-27 | Aerpio Pharmaceuticals, Inc. | Methods for treating vascular leak syndrome and cancer |
US12043664B2 (en) | 2011-10-13 | 2024-07-23 | EyePoint Pharmaceuticals, Inc. | Methods for treating vascular leak syndrome and cancer |
EP3424530A1 (fr) | 2013-03-15 | 2019-01-09 | Zyngenia, Inc. | Complexes multispécifiques monovalents et multivalents et leurs utilisations |
US10220048B2 (en) | 2013-03-15 | 2019-03-05 | Aerpio Therapeutics, Inc. | Compositions and methods for treating ocular diseases |
US9994560B2 (en) | 2014-03-14 | 2018-06-12 | Aerpio Therapeutics, Inc. | HPTP-β inhibitors |
US10858354B2 (en) | 2014-03-14 | 2020-12-08 | Aerpio Pharmaceuticals, Inc. | HPTP-Beta inhibitors |
US11666558B2 (en) | 2015-09-23 | 2023-06-06 | EyePoint Pharmaceuticals, Inc. | Methods of treating intraocular pressure with activators of Tie-2 |
US10952992B2 (en) | 2015-09-23 | 2021-03-23 | Aerpio Pharmaceuticals, Inc. | Methods of treating intraocular pressure with activators of Tie-2 |
US12171751B2 (en) | 2015-09-23 | 2024-12-24 | EyePoint Pharmaceuticals, Inc. | Methods of treating intraocular pressure with activators of Tie-2 |
US11253502B2 (en) | 2019-04-29 | 2022-02-22 | EyePoint Pharmaceuticals, Inc. | Tie-2 activators targeting the Schlemm's canal |
US12064420B2 (en) | 2019-04-29 | 2024-08-20 | EyePoint Pharmaceuticals, Inc. | Tie-2 activators targeting the Schlemm's canal |
Also Published As
Publication number | Publication date |
---|---|
AU5154098A (en) | 1998-05-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO1998018914A1 (fr) | Recepteur tie2 soluble | |
Lin et al. | Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor | |
AU2006287228B2 (en) | Methods for using and identifying modulators of Delta-like 4 | |
Lin et al. | Inhibition of tumor angiogenesis using a soluble receptor establishes a role for Tie2 in pathologic vascular growth. | |
KR100336452B1 (ko) | 앤지오스타틴및맥관형성억제를위한이의사용방법 | |
US8153585B2 (en) | Peptide antagonists of vascular endothelial growth factor and methods of use thereof | |
JP3787157B2 (ja) | アンジオスタチンフラグメント、アンジオスタチン凝集体および使用方法 | |
JP2001506506A (ja) | アンジオスタチンフラグメントおよび使用方法 | |
JP4404479B2 (ja) | 血管内皮増殖因子の可溶性インヒビターおよびその使用 | |
JP4312955B2 (ja) | 血管内皮増殖因子のペプチドアンタゴニスト | |
EP1196186A2 (fr) | Stimulation ou inhibition de l'angiogenese et de la cardiovascularisation avec des homologues de ligands et de recepteurs du facteur de necrose tumorale | |
KR100682666B1 (ko) | 항혈관형성 단백질 및 이들을 사용하는 방법 | |
JP2006213724A (ja) | アンジオスタチンおよび血管形成の抑制におけるその使用 | |
US6498144B1 (en) | Use of scatter factor to enhance angiogenesis | |
KR20010020342A (ko) | 종양치료용 맥관형성 길항제의 아데노바이러스-매개 종양내 전달방법 | |
JP3684371B2 (ja) | 内皮細胞増殖阻害剤およびその使用法 | |
CA2312287A1 (fr) | Procede de production de proteines anti-angiogeniques, dont l'endostatine, l'angiostatine ou la restine, au moyen d'un systeme d'expression de la levure pichia | |
JP2003517007A (ja) | 内皮細胞増殖阻害組成物および方法 | |
US6797488B1 (en) | Methods of producing anti-angiogenic proteins | |
US6133416A (en) | Inhibition of cell growth by an anti-proliferative factor | |
CA2411056C (fr) | Compositions pharmaceutiques renfermant le gene de la prostacyline synthase | |
AU2001265082A1 (en) | Thrombospondin-1 type 1 repeat polypeptides | |
US7622443B2 (en) | Method for inhibiting pro-angiogenic activities of endothelial cells selectively at a site of neoangiogenesis in a mammal by administration of the extracellular domain of D1-1 polypeptides | |
KR20010092800A (ko) | 펩타이드 | |
CA2477867A1 (fr) | Peptides, adn, arn et composes pour inhiber ou amorcer l'activite de l'adrenomedulline et leur utilisation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |