CA2331370A1 - Protein oligomer compositions comprising endostatin protein and methods of using the same - Google Patents
Protein oligomer compositions comprising endostatin protein and methods of using the same Download PDFInfo
- Publication number
- CA2331370A1 CA2331370A1 CA002331370A CA2331370A CA2331370A1 CA 2331370 A1 CA2331370 A1 CA 2331370A1 CA 002331370 A CA002331370 A CA 002331370A CA 2331370 A CA2331370 A CA 2331370A CA 2331370 A1 CA2331370 A1 CA 2331370A1
- Authority
- CA
- Canada
- Prior art keywords
- endostatin
- oligomer
- protein
- zinc
- oligomers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010079505 Endostatins Proteins 0.000 title claims abstract description 251
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 90
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 54
- 102100031162 Collagen alpha-1(XVIII) chain Human genes 0.000 title claims abstract 16
- 239000000203 mixture Substances 0.000 title abstract description 23
- 239000011701 zinc Substances 0.000 claims abstract description 41
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229910052725 zinc Inorganic materials 0.000 claims abstract description 40
- 239000013638 trimer Substances 0.000 claims abstract description 19
- 208000005623 Carcinogenesis Diseases 0.000 claims abstract description 5
- 230000036952 cancer formation Effects 0.000 claims abstract description 5
- 231100000504 carcinogenesis Toxicity 0.000 claims abstract description 5
- 239000000178 monomer Substances 0.000 claims description 66
- 239000000539 dimer Substances 0.000 claims description 42
- 230000000694 effects Effects 0.000 claims description 30
- 239000012634 fragment Substances 0.000 claims description 22
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 claims description 18
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 claims description 18
- 210000002889 endothelial cell Anatomy 0.000 claims description 15
- 229910052751 metal Inorganic materials 0.000 claims description 15
- 239000002184 metal Substances 0.000 claims description 15
- 108010001463 Collagen Type XVIII Proteins 0.000 claims description 12
- 102000047200 Collagen Type XVIII Human genes 0.000 claims description 12
- 238000001502 gel electrophoresis Methods 0.000 claims description 10
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 108020001507 fusion proteins Proteins 0.000 claims description 8
- 102000037865 fusion proteins Human genes 0.000 claims description 8
- 230000002622 anti-tumorigenesis Effects 0.000 claims description 6
- 230000015916 branching morphogenesis of a tube Effects 0.000 claims description 3
- 230000000259 anti-tumor effect Effects 0.000 claims description 2
- 230000000447 dimerizing effect Effects 0.000 claims 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 18
- 210000005239 tubule Anatomy 0.000 abstract description 11
- 229910021645 metal ion Inorganic materials 0.000 abstract description 7
- 239000013636 protein dimer Substances 0.000 abstract 1
- 102400001047 Endostatin Human genes 0.000 description 220
- 235000018102 proteins Nutrition 0.000 description 54
- 206010028980 Neoplasm Diseases 0.000 description 36
- 210000004027 cell Anatomy 0.000 description 34
- 230000033115 angiogenesis Effects 0.000 description 31
- 101500026378 Homo sapiens Endostatin Proteins 0.000 description 21
- 201000010099 disease Diseases 0.000 description 21
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 21
- 235000001014 amino acid Nutrition 0.000 description 19
- 150000001413 amino acids Chemical class 0.000 description 16
- 238000003556 assay Methods 0.000 description 14
- 239000013078 crystal Substances 0.000 description 13
- 230000001419 dependent effect Effects 0.000 description 13
- 210000001519 tissue Anatomy 0.000 description 13
- 238000009472 formulation Methods 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 230000027455 binding Effects 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 9
- 239000003446 ligand Substances 0.000 description 9
- 150000007523 nucleic acids Chemical group 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 108020003175 receptors Proteins 0.000 description 9
- 102000005962 receptors Human genes 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 230000004572 zinc-binding Effects 0.000 description 9
- 102100029727 Enteropeptidase Human genes 0.000 description 8
- 108010013369 Enteropeptidase Proteins 0.000 description 8
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 230000001772 anti-angiogenic effect Effects 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 108010035532 Collagen Proteins 0.000 description 7
- 102000008186 Collagen Human genes 0.000 description 7
- 206010027476 Metastases Diseases 0.000 description 7
- 238000007792 addition Methods 0.000 description 7
- 229920001436 collagen Polymers 0.000 description 7
- 238000004132 cross linking Methods 0.000 description 7
- 230000003511 endothelial effect Effects 0.000 description 7
- 235000014304 histidine Nutrition 0.000 description 7
- 150000002411 histidines Chemical class 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 108010079709 Angiostatins Proteins 0.000 description 6
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 6
- 230000002491 angiogenic effect Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000000470 constituent Substances 0.000 description 6
- 238000006471 dimerization reaction Methods 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 238000006384 oligomerization reaction Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 210000002469 basement membrane Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 235000004554 glutamine Nutrition 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 208000037819 metastatic cancer Diseases 0.000 description 5
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 5
- 206010061289 metastatic neoplasm Diseases 0.000 description 5
- 230000008521 reorganization Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 102400000068 Angiostatin Human genes 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- 102100032813 Hepatocyte growth factor-like protein Human genes 0.000 description 4
- 101710086591 Hepatocyte growth factor-like protein Proteins 0.000 description 4
- 102000002265 Human Growth Hormone Human genes 0.000 description 4
- 108010000521 Human Growth Hormone Proteins 0.000 description 4
- 239000000854 Human Growth Hormone Substances 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 206010029113 Neovascularisation Diseases 0.000 description 4
- 206010038933 Retinopathy of prematurity Diseases 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 235000003704 aspartic acid Nutrition 0.000 description 4
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 210000004292 cytoskeleton Anatomy 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 108010082117 matrigel Proteins 0.000 description 4
- 235000008729 phenylalanine Nutrition 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- 102100025698 Cytosolic carboxypeptidase 4 Human genes 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101000932590 Homo sapiens Cytosolic carboxypeptidase 4 Proteins 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 101001033003 Mus musculus Granzyme F Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 3
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 3
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 3
- 239000004037 angiogenesis inhibitor Substances 0.000 description 3
- YXVFYQXJAXKLAK-UHFFFAOYSA-N biphenyl-4-ol Chemical group C1=CC(O)=CC=C1C1=CC=CC=C1 YXVFYQXJAXKLAK-UHFFFAOYSA-N 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000001493 electron microscopy Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 230000000921 morphogenic effect Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 210000003606 umbilical vein Anatomy 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- VEEGZPWAAPPXRB-BJMVGYQFSA-N (3e)-3-(1h-imidazol-5-ylmethylidene)-1h-indol-2-one Chemical compound O=C1NC2=CC=CC=C2\C1=C/C1=CN=CN1 VEEGZPWAAPPXRB-BJMVGYQFSA-N 0.000 description 2
- 206010000050 Abdominal adhesions Diseases 0.000 description 2
- 208000003120 Angiofibroma Diseases 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
- 208000003732 Cat-scratch disease Diseases 0.000 description 2
- 208000032544 Cicatrix Diseases 0.000 description 2
- 206010011017 Corneal graft rejection Diseases 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 206010012689 Diabetic retinopathy Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 208000010412 Glaucoma Diseases 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 208000002260 Keloid Diseases 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 201000004404 Neurofibroma Diseases 0.000 description 2
- 102400001048 Non-collagenous domain 1 Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 201000004681 Psoriasis Diseases 0.000 description 2
- 206010037649 Pyogenic granuloma Diseases 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 206010039710 Scleroderma Diseases 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 206010043189 Telangiectasia Diseases 0.000 description 2
- 108060008245 Thrombospondin Proteins 0.000 description 2
- 102000002938 Thrombospondin Human genes 0.000 description 2
- 241000390203 Trachoma Species 0.000 description 2
- 208000025865 Ulcer Diseases 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 208000004064 acoustic neuroma Diseases 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- -1 alto-phycocyanin Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 229920002988 biodegradable polymer Polymers 0.000 description 2
- 239000004621 biodegradable polymer Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000001043 capillary endothelial cell Anatomy 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000005059 dormancy Effects 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229940126864 fibroblast growth factor Drugs 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000005469 granulation Methods 0.000 description 2
- 230000003179 granulation Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 201000011066 hemangioma Diseases 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000001969 hypertrophic effect Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000007917 intracranial administration Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 210000001117 keloid Anatomy 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 208000002780 macular degeneration Diseases 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 201000003142 neovascular glaucoma Diseases 0.000 description 2
- 230000036963 noncompetitive effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 150000002994 phenylalanines Chemical class 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000001147 pulmonary artery Anatomy 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 125000006853 reporter group Chemical group 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 231100000241 scar Toxicity 0.000 description 2
- 230000037387 scars Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 210000003518 stress fiber Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 208000009056 telangiectasis Diseases 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 206010044325 trachoma Diseases 0.000 description 2
- 238000005829 trimerization reaction Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 231100000397 ulcer Toxicity 0.000 description 2
- 210000001644 umbilical artery Anatomy 0.000 description 2
- 238000012800 visualization Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- LQZFQLVBZRVDLJ-DKWTVANSSA-N (2s)-2-aminobutanedioic acid;zinc Chemical compound [Zn].OC(=O)[C@@H](N)CC(O)=O LQZFQLVBZRVDLJ-DKWTVANSSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 101710112984 20 kDa protein Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000012936 Angiostatins Human genes 0.000 description 1
- 108020005098 Anticodon Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000012756 BrdU staining Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- BVTJGGGYKAMDBN-UHFFFAOYSA-N Dioxetane Chemical class C1COO1 BVTJGGGYKAMDBN-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000027430 HGF receptors Human genes 0.000 description 1
- 108091008603 HGF receptors Proteins 0.000 description 1
- 208000002125 Hemangioendothelioma Diseases 0.000 description 1
- 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 1
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 1
- 101500026377 Homo sapiens Non-collagenous domain 1 Proteins 0.000 description 1
- 101001123448 Homo sapiens Prolactin receptor Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101710159910 Movement protein Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101500026380 Mus musculus Endostatin Proteins 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 208000003788 Neoplasm Micrometastasis Diseases 0.000 description 1
- 101800000353 Non-collagenous domain 1 Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 108010002519 Prolactin Receptors Proteins 0.000 description 1
- 102100029000 Prolactin receptor Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710150114 Protein rep Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 101710152114 Replication protein Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101710137510 Saimiri transformation-associated protein Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 102000003800 Selectins Human genes 0.000 description 1
- 108090000184 Selectins Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 description 1
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 1
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 1
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 1
- 101710090398 Viral interleukin-10 homolog Proteins 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000007801 affinity label Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 150000001295 alanines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 238000011122 anti-angiogenic therapy Methods 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000032341 cell morphogenesis Effects 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 238000002967 competitive immunoassay Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012866 crystallographic experiment Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000003436 cytoskeletal effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000010339 dilation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000037765 diseases and disorders Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- QNDQILQPPKQROV-UHFFFAOYSA-N dizinc Chemical compound [Zn]=[Zn] QNDQILQPPKQROV-UHFFFAOYSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000007421 fluorometric assay Methods 0.000 description 1
- 238000001730 gamma-ray spectroscopy Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 150000002309 glutamines Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000012309 immunohistochemistry technique Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000000185 intracerebroventricular administration Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000006510 metastatic growth Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000002135 phase contrast microscopy Methods 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 210000004910 pleural fluid Anatomy 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000009290 primary effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 102220005395 rs33991223 Human genes 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000003345 scintillation counting Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 229940006486 zinc cation Drugs 0.000 description 1
- 108010088577 zinc-binding protein Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Protein oligomer compositions comprising endostatin protein are provided as well as methods of using the protein oligomer compositions to disrupt tubule formation and inhibit tumorigenesis. The compositions of the present invention constitute a new class of scatter factors and specifically include endostatin protein dimers and trimers, and optionally include metal ions such as zinc.
Description
WO 99/62944 PCTJt1S99/12278 PROTEIN OLIGOMER COMPOSITIONS COMPRISING
ENDOSTATIN PROTEIN AND METHODS
OF USING THE SAME
Cross Reference to Related Applications This application claims priority to provisional application Serial No. 60/087,890 filed June 3, 1998; provisional application Serial No. 60J092,393 filed July 10, 1998; and provisional application Serial No. 60/098,790 filed September l, 1998. Each of the above-referenced applications is incorporated herein in its entirety.
Field of the Invention The present invention relates to the fields of oncology, angiogenesis and morphogenesis and more particularly to novel protein oligomers comprising endostatin protein. The endostatin oligomers are useful fox inhibiting endothelial cell tubule formation, regulating cellular morphogenesis, and treating metastatic cancers and angiogenesis-dependent diseases.
Background of the Invention Several lines of direct evidence now suggest that the growth and persistence of solid tumors as well as their metastases to distant organs is critically dependent upon angiogenesis, or the recruitment of new blood vessels (Folkman, 1989; Hori et al., WO 99/b2944 PCT/US99/12278 1991; Kim et al., 1993; Millauer et al., 1994). Angiogenesis not only provides the increased nutrients and pathways for the removal of waste needed for the expansion of the tumor, but it also facilitates tumor metastasis by providing a route for tumor cells to leave the primary site and enter the bloodstream (letter, 1998). In particular, angiogenesis increases the entry of tumor cells into the bloodstream by providing an increased density of immature, highly permeable blood vessels that have thinner basement membranes and fewer intracellular junction complexes than normal mature vessels (letter, 1998).
It is postulated that the angiogenic phenotype is the result of a net balance between both positive and negative regulators of neovascularization (Good et al., 1990; O'Reilly et al., 1994;
Parangi et al., 1996; Rastinejad et aL, 1989). Tumors themselves, along with other accessory host cells such as macrophages, mast cells and lymphocytes; stimulate angiogenesis by up-regulating their production of a variety of angiogenic factors, including the fibroblast growth factor (FGF). (Kandel et al., 1991 ) and vascular endothelial cell growth factor/vascular permeability factor (VEGF/VPF) (letter, 1998). However, many malignant tumors also generate inhibitors of angiogenesis, including angiostatin protein and thrombospondin (Chen et al., 1995; Good et al., 1990; O'Reilly et al., 1994; U.S. Patent No. 5,639,725).
Several angiogenic and anti-angiogenic proteins are stored as inactive precursors in the blood or basement membranes (Hanahan, 1996). One example of such an inactive precursor is endostatin protein. Endostatin protein is an approximately 20 kDa C-terminal globular domain of the collagen-like protein, collagen XVIII (c18) which is localized in the basement membrane around blood vessels {Oh et al., 1994). Endostatin is stored in vivo as the C-terminal portion of the c 18 protein (Rehn et al., 1994;
Muragaki et al., 1995) and fragments of c 18 longer than endostatin are believed not to inhibit endothelial cell proliferation (O'Reilly et al., 1997). Endostatin was first isolated from a hemangioendothelioma cell line for its ability to inhibit the WO 99!62944 PCT/US99I12278 ' 3 proliferation of capillary endothelial cells as described in U.S.
Patent Number 5,854,205, which is hereby incorporated by reference.
Although it is known that endostatin is stored as an inactive precursor, it is not known how endostatin is activated. Previous x-ray crystallographic studies indicated that endostatin does not contain the characteristic Ca2+ binding sites that are active in selectins, a similar group of molecules {Hohenester et al., 1998).
These same studies indicated that no metal ions were associated with human endostatin.
Metal ions are often involved in the biologic activity of proteins. In particular, the zinc cation is a critical component of many proteins and plays a key role in a host of biological processes (Coleman, 1992; O'Reilly et al, 1996, Vallee et al.
1990). In most cases, zinc is directly involved in the catalytic activity, however structural roles for zinc have also been described. For example, zinc is involved in the dimerization of human growth hormone and increases the affinity of human growth hormone for the prolactin receptor by approximately 8,000 fold {Cunningham et al., 1990; Cunningham et al., 1991 ).
Summary of the Lnvention The present invention includes protein oligomers comprising more than one endostatin protein monomers, wherein the oligomer has scatter factor activity. Endostatin protein is a carboxy-terminal region fragment of collagen XVIII having a molecular weight of approximately 20 kDa as determined by reducing gel electrophoresis and I8 kDa as determined by non-reducing gel electrophoresis. In one embodiment of the present invention, the protein oligomer comprises a dirner of endostatin monomers. In an alternate embodiment, the protein oligomer comprises more than one NCI region fragment of collagen XVIII
having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC 1 fragment contains an endostatin monomer. In a preferred embodiment, the protein oligomer comprising more than one NC 1 region fragment is a trimer. The protein oligomers of the present invention may optionally comprise a metal component, which is preferably zinc.
The novel protein oligomers described herein constitute a novel class of scatter factors. The protein oligomers of the present invention have anti-tubulogenic effects and induce reorganization of the actin cytoskeleton, destruction of tubular lumens, and elongation of cells. The novel protein oligomers axe also anti-tumorigenic and anti-angiogenic.
Also included in the present invention are methods of using the protein oligomers comprising endostatin protein. The protein oligomers described herein may be used to inhibit tubulogenesis and tumorigenesis.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
Brief Description of the Figures Figure 1. Structure of Hurrcan Endostatin 13 strands are labeled in sequential order A-P, a helices are denoted, and zinc is shown as a sphere.
Figure 2. The N-terminal Loop and Zinc Binding Site of Human Endostatin The zinc (black circle) ligands histidines 1, 3, and 1 i and aspartic acid 76, as well as the second shell of interactions that position the zinc ligands, glutamic acid 175, residue 11 carbonyl oxygen, and arginine 4 from the N-terminal loop of an adjacent molecule in a dimer are shown as ball and stick models ' 5 Figure 3A-B. A Zinc Dependent Dimer in Human Endostatin Crystals Figure 3A. The zinc (black sphere) site N-terminal loops of two monomers contact across a central dyad axis. Glutamine 7, phenylalanine 6, and arginine 5 of the loop project from one monomer to the next. Also shown are two phenylalanine rings, residues 31 and 34, that project from an endostatin oc helix and form another dimeric contact in the crystal.
Figure 3B. Contacts in the interface of the dimer seen in IO crystals of human endostatin. Zinc (black sphere) ligands have open bonds, interface residues have solid bonds. The path of the polypeptide chains of the two monomers are shown as tubes. The solvent accessible surface buried in this dimer interface is 403 ~2 (probe size = 1.4 ~.) per monomer.
Figure 4. Oligomeric Endostatins Exhibit Scatter Factor Activity HUVEC tubules on matrigel were pre-formed for 16 hours and treated with human endostatin monomer, human Fc endostatin dimer, human Fc dimers, human Fc-endostatin(C7) dimers, human endostatin(C7) dimers, human endostatin/NC1 trimers, human Fc collagen 15 dimers, or Fc angiostatin dimers.
Oniy endostatin dimers and trimers exhibited scatter activity and inhibited tubule formation.
Detailed Description The present invention provides a new class of scatter factors comprising endostatin oligomers. Oligomers in this new class of scatter factors act as "anti-matrix" scatter factors and are capable of inhibiting endothelial tubule assembly evoked by the presence of extracellular matrix proteins. The endostatin oligomers are additionally anti-angiogenic and anti-tumorigenic.
Although the endostatin oligomers have been defined functionally, it is to be understood that this functional definition in no way limits the bioactivity of the endostatin oligomers.
WO 99/62944 PCTfUS99/I2278 The protein oligomers of _one embodiment of the present invention comprise a plurality of endostatin monomers, wherein the endostatin monomers are approximately 20 kDa proteins as determined under reduced gel electrophoresis or approximately 18 kDa as determined under non-reduced gel electrophoresis, and are characterized by their ability to inhibit proliferating cultured endothelial cells. The endostatin oligomers are carboxy-region fragments of collagen-like molecules such as collagen XVIII, and may be derived from any mammal. In a preferred embodiment, the endostatin monomer begins at approximately amino acid position 1132 of murine collagen XVIII, and correlates to the human endostatin fragment of SEQ ID NO:1 shown below.
HSHRDFQPVLHLVALNSFLSGGMRGIRGADFQCFQQARAVG
LAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSW
EALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKS V WHGSD
PNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASC
HHAYIVLCIENSFMTAS
As described below, amino acid substitutions may occur in the sequence of endostatin which still yield a functional endostatin protein. For example, when the above gene sequence is recombinantly expressed, an observable doublet of protein results, both versions of which are functional endostatin proteins.
In addition to the above endostatin protein, the following endostatin variant occurs, which is the former protein minus the first four amino acids: This demonstrates the variability of functional endostatin protein molecules. Therefore, in an alternate embodiment, the endostatin monomer correlates to the human endostatin fragment of SEQ ID N0:2 shown below.
DFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAG
TFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEAL
FSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNG
RRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA
YIVLCIENSFMTAS
The terms "endostatin" and "endostatin monomer" are synonymous and include naturally occurring, recombinant, or synthetic endostatin proteins that contain conservative, or "silent"
amino acid substitutions, deletions and additions, yet upon oligomerization retain scatter factor activity. The term "scatter factor activity" refers to the disruption of endothelial tubule formation as determined by a matrigel tube-formation assay. In a preferred embodiment, the endostatin monomers are modified at the seventh amino acid such that glutamine is replaced with cysteine. Replacement of glutamine with cysteine facilitates dimerization or oligomerization between endostatin monomers.
The term "endostatin monomer" also includes shortened proteins wherein one or more amino acid is removed from either or both ends of an endostatin monomer, or from an internal region of the protein, yet upon oligomerization retain scatter factor activity.
The term "endostatin monomer" also includes lengthened proteins or peptides wherein one or more amino acids is added to either or both ends of an endostatin monomer, or to an internal location, yet upon oligomerization retain scatter factor activity. One example of such a modification is the addition of tyrosine to the first position. Tyrosine labeled molecules may be further labeled with lasiodine for use in assays. Labeling with other radioisotopes or chemicals such as ricin may also be useful in providing a molecular tool for destroying the target cells containing endostatin oligomer receptors. Endostatin monomers can also be recombinantly fused to other proteins or peptides, such as a Fc portion of an antibody as described in the Examples below.
Additionally, silent substitutions of amino acids, are well known in the art and are intended to fall within the scope of the appended claims. Silent substitutions occur when the replacement of an amino acid with a structurally or chemically similar amino acid does not significantly alter the structure, conformation or , 8 activity of the protein. Also included in the definition of the term "endostatin monomer" are modifications of the protein, its subunits and peptide fragments. Such modifications include substitutions of naturally occurring amino acids at specific sites with other molecules, including but not limited to naturally and -non-naturally occurring amino acids. Such substitutions may modify the bioactivity of endostatin oligomers, such as by increasing or decreasing the scatter factor activity, and produce biological or pharmacological agonists or antagonists.
l0 In one embodiment, the protein oligomers are endostatin dimers. The present invention includes a novel class of scatter factors that includes endostatin dimers. The endostatin dimers of the present invention progressively disperse established tubes into constituent cells as early as 2-3 hours and without obvious toxicity. During the endostatin dimer induced scatter, dramatic reorganization of the cytoskeleton can be observed along with destruction of tubular lumens and cellular elongation. The endostatin oligomers differ from the previously described Hepatocyte Growth Factor/ Scatter Factor (HGF/SF) class of 2o scatter factors. In contrast to the HGF/SF class of scatter factors that promote tube formation, the endostatin oligomers of the present invention have anti-tubulogenic effects. The endostatin oligomers also affect different types of cells as compared to the HGF/SF class of scatter factors. In addition to scatter activity, the endostatin dimers of the present invention are also capable of anti-tumorigenic and anti-angiogenic activity when bound to a metal such as zinc.
In an alternate embodiment, the invention provides protein oligomers comprising a plurality of endostatin/NCl proteins. The endostatin/NC 1 proteins of the present invention are approximately 38 kDa C-terminal region fragments of collagen XVIII that each contain the approximately 20 kDa endostatin proteins described above. The present invention describes for the first time that endostatin/NC 1 trimers are included in the new class of scatter factors described above. Endostatin/NC 1 trimers induce scattering of endothelial cell tubular structures as well as other cellular morphogenic changes in a manner similar to endostatin dimers. The anti-tubulogenic activity of the endostatin/NC 1 trimers can also be inhibited by endostatin monomers.
In another embodiment, the protein oligomers described above comprise Pndostatin monomers that are fusion proteins.
The endostatin fusion proteins may comprise endostatin and anti-angiogenic molecules, angiogenic molecules, and/or molecules that facilitate dimerization of the endostatin monomers. In a preferred embodiment, the endostatin fusion proteins comprise endostatin and the Fc portion of an antibody; wherein the Fc portion of the antibody promotes dimerization. The Fc portion may be derived from the IgG, IgE, IgA or IgM isotype, however, the preferred isotype is IgG.
In a further embodiment, the protein oligomers, dimers and monomers are bound to a metal ion, preferably a zinc ion.
The, invention provides for the first time that endostatin contains a zinc binding site and requires metal binding for activity. While prior attempts to study crystallized endostatin resulted in disordered residues and inaccurate results, the inventors have expressed endostatin monomers in such a manner that the residues are not disordered and have identified a zinc binding domain on endostatin. In the present invention, human endostatin has been expressed as a secreted protein in a murine myeloma cell line as a chimera with the Fc domain of IgG-1 and released from the Fc portion by enterokinase digestion (as more specifically described below). Additionally human endostatin crystals were grown at a higher pH where the N-terminal histidines would not be charged.
These modifications of prior art methods have allowed the inventors to accurately identify endostatin as a zinc binding protein.
Using the above methods, the inventors discovered that the zinc binding site of human endostatin is tetrahedral with three zinc ligands from the N-terminal loop, histidines 1, 3, and l l, and WO 99162944 PCTlUS99/12278 ' 10 a fourth ligand, aspartic acid 76, from the loop between the E and F 13-strands (Fig. 1 and Fig. 2). The inventors also describe for the first time zinc dependent dimeric contacts between endostatin monomers in human endostatin crystals. Based upon these findings, the present invention provides novel endostatin compositions bound to zinc wherein zinc is essential to the anti-tumorigenic activity of the endostatin compositions, and methods of using these compositions. In a preferred embodiment, the Fc-endostatin dimers are bound to zinc. In another preferred embodiment, NC 1/endostatin trimers are bound to zinc.
The present invention also encompasses methods of activating endostatin in vitro or in vivo comprising the binding of one or more metal ions to endostatin oligomers. In a preferred embodiment, the metal ion is zinc, and in a further preferred embodiment, one zinc molecule is bound per endostatin monomer. Activation of endostatin includes increased anti-angiogenic activity, increased tumorigenic activity and/or increased scatter factor activity. In one embodiment, zinc binding causes dimerization of endostatin monomers and consequently activates endostatin. Also included in the invention are methods of forming endostatin oligomers by the addition of a metal ion, preferably a zinc ion.
Typically, the isolated, endostatin oligomers of the invention are at least about 80% pure, usually at least about 90%, and preferably at Ieast about 95 % as measured by band intensity on a silver stained gel. The phrases "isolated", "biologically pure"
or "substantially pure" refer to material which is essentially free from at least some of the components which normally accompany it as found in its native state. Other important terms that are used herein are defined as follows. The terms "a", "an" and "the" as used herein are defined to mean one or more and include the plural unless the context is inappropriate. "Anti-tumor activity"
and "anti-tumorigenic" refer to the ability of an endostatin oligomer to regress a tumor. "Regression" refers to the reduction of tumor mass or size. The term "anti-angiogenic" refers to inhibition of vascularization. Additionally, "substantial sequence homology" means at least approximately 70% homology between a known amino acid residue sequence in an endostatin oligomer, preferably at least approximately 80% homology, more preferably at least approximately 90% homology. Homology can be determined by sequence identity, or by using a well-known computer program, such as DNA Star or GeneJockey, on default setting parameters.
The endostatin and NC 1 monomers that constitute portions of the protein oligomers described above may be isolated from body fluids and tissues including, but not limited to, serum, urine and ascites fluid. Endostatin monomers and NC 1 may also be produced from recombinant sources, genetically altered cells implanted into animals, tumors, cell cultures and other sources.
Recombinant techniques include gene amplification from DNA
sources using amplification techniques such as the polymerase chain reaction (PCR), and gene amplification from RNA sources using amplification techniques such as reverse transcriptase/PCR.
The endostatin monomers may be produced by polypeptide synthesis, or derived by ~n vitro enzymatic catalysis of larger, encompassing polypeptides such as collagen XVIII to yield active endostatin monomers. In a preferred embodiment, the endostatin monomers of the present invention are produced from a recombinant expression system, wherein the cell is Pichia pastoriS.
The endostatin oligomers of the present invention have several uses. The endostatin oligomers are particularly useful for inhibiting endothelial cell tubule formation and for the study of morphogenesis. Endostatin oligomers, and active fragments thereof, are also useful for treating metastatic cancers and tumors as well as angiogenesis-related cancers and diseases.
For example, the endostatin oligomers may be employed to treat metastatic and angiogenesis-dependent cancers and other angiogenesis-related diseases. One example of metastatic disease is metastatic cancer. Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, fox example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; and wound granulation. The endostatin oligomers of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids.
They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helobacter pylori}.
The endostatin oligomers may also be used to develop affinity columns for isolating antibodies directed toward the endostatin oligomers. Those antibodies may be isolated and purified, followed by amino acid sequencing. Also, polypeptides that bind to endostatin oligomers with high specificity and avidity may be labeled with a label or reporter group and employed for visualization and quantitation in the assays described herein using detection techniques such as autoradiographic and membrane binding techniques. The reporter group or label is commonly a fluorescent or radioactive group or an enzyme. Such applications provide important diagnostic and research tools.
Endostatin oligomers can also be employed to develop affinity columns for isolation of endostatin oligomer receptors.
Isolation and purification of such receptors may be followed by amino acid sequencing. Using this information, the gene or genes coding for the receptors can be identified and isolated. Next, cloned nucleic acid sequences may be developed fox insertion into vectors capable of expressing the receptors.
Applicants' invention also encompasses recombinant expression systems wherein nucleic acids encoding the protein constituents of the endostatin oligomers described herein are contained in a single cell. The nucleic acids encoding the oligomeric constituents may be in a single vector or multiple vectors. In a preferred embodiment, the recombinant expression system is Pichia pastoris. The biologically active endostatin oligomers and nucleic acid sequences corresponding to the proteins are useful for modulating endothelial processes in vivo, and for diagnosing and treating endothelial cell-related diseases, for example by gene therapy.
Nucleic acid sequences that correspond to, and code for, the protein constituents of endostatin oligomers can be prepared based upon the knowledge of the amino acid sequence, and the art recognized correspondence between codons (sequences of three nucleic acid bases), and amino acids. Because of the degeneracy of the genetic code, wherein the third base in a codon may vary, yet still code for the same amino acid, many different possible coding nucleic acid sequences are derivable for any particular protein or peptide fragment. Alternatively, the nucleic acid sequence may be derived from a gene bank using oligonucleotides probes designed based on the N-terminal amino acid sequence and well known techniques for cloning genetic material. Nucleic acid sequences are synthesized using automated systems well known in the art.
Either the entire sequence may be synthesized or a series of smaller oligonucleotides are made and subsequently ligated together to yield the full length sequence. The genes encoding constituents of endostatin oligorners may also be isolated from cells or tissue that express high levels of endostatin monomers or NC 1 by ( 1 ) isolating messenger RNA from the tissue, (2) using reverse transcriptase to generate the corresponding DNA
sequence and then (3) using PCR with the appropriate primers to amplify the DNA sequence coding for the active sequence.
w0 99/62944 PCT/US99/12278 ' 14 The isolated, recombinant or synthetic endostatin oligomers described herein are useful for generating antibodies specific for the endostatin oligomers. These antibodies that specifically bind to the endostatin oligomers can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect or quantify the endostatin oligomers in a body fluid or tissue. Results from these tests can be used to diagnose or predict the occurrence or recurrence of a cancer and other angiogenesis mediated diseases. Additionally, passive antibody therapy using antibodies that specifically bind endostatin oligomers can be employed to modulate morphogenic processes such as metastatic cancer as well as angiogenesis-dependent processes such as reproduction, development, wound healing, tissue repair, and angiogenesis-dependent diseases. Also, antisera directed to the Fab I5 regions of endostatin oligomer antibodies can be administered to block the ability of endogenous endostatin oligomer antisera to bind endostatin oligomers.
Antibodies specific for endostatin oligomers may be either polyclonal or monoclonal, and are made according to techniques and protocols well known in the art. The preferred method of making monoclonal antibodies is a modified version of the method of Kearney et al. ( 1979), which is incorporated by reference herein. The monoclonal antibodies are utilized in well known immunoassay formats, such as competitive and non-competitive immunoassays, including ELISA, sandwich immunoassays and radioimmunoassays (RIAs), to determine the presence or absence of the endostatin oligomers of the present invention in body fluids and tissues. Examples of body fluids include, but are not limited to, blood, serum, synovial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterine fluid, saliva, mucus and vitreous humor.
Polyclonal antisera are also raised using established techniques known to those skilled in the art. For example, polyclonal antisera may be raised in mice, rabbits, rats, goats, sheep, guinea pigs, chickens, and other animals, most preferably WO 99/b2944 PCT/US99/12278 mice and rabbits by administering the antigen to the animals.
Isolated, recombinant or synthetic endostatin oligomers conjugated to a carrier molecule such as bovine serum albumin, may be combined with an adjuvant mixture, emulsified and 5 injected subcutaneously at multiple sites on the back, neck, flanks, and sometimes in the footpads of the animals. Booster injections are made at regular intervals, such as every two to four weeks.
Blood samples are obtained by venipuncture, for example using the marginal ear veins after dilation, approximately seven to ten 10 days after each injection. The blood samples are allowed to clot and are centrifuged, and the serum removed, aliquoted, and stored under refrigeration for immediate use or frozen for subsequent analysis.
Techniques for the production of single chain antibodies 15 are known to those skilled in the art and described in U.S. Patent No. 4,946,778 and can be used to produce single chain antibodies to the endostatin oligomers described herein. Bispecific antibodies have two antigen binding domains wherein each domain is directed against a different epitope. Phage display technology may be used to select antibody genes having binding activities for endostatin oligomers from PCR-amplified v genes of lymphocytes from naive libraries or humans screened for having antibodies to the endostatin oligomers.
When labeled with a detectable biomolecule or chemical, the endostatin oligomers and antibodies described above are useful for purposes such as in vivo and in vitro diagnostics and laboratory research using the methods and assays described below. Various types of labels and methods of conjugating the labels to the endostatin oligomers and antibodies are well known to those skilled in the art. Several specific labels are set forth below.
For example, the endostatin oligomers and antibodies are conjugated to a radiolabel such as, but not restricted to, 32P, 3H, 14C, 355, 1251, or 131I. Detection of a label can be by methods such as scintillation counting,, gamma ray spectrometry o r autoradiography.
Bioluminescent labels, such as derivatives of firefly Iuciferin, are also useful. The bioluminescent substance is covalently bound to the endostatin oligomer or antibody by conventional methods, and the labeled endostatin oligomer or antibody is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light.
Fluorogens may also be used as labels. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, alto-phycocyanin, phycocyanin; rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector.
The endostatin oligomers and antibodies can alternatively be labeled with a chromogen to provide an enzyme or affinity label. For example, the endostatin oligomer or antibody can be biotinylated so that it can be utilized in a biotin-avidin reaction; which may also be coupled to a label such as an enzyme or fluorogen. Alternatively, the endostatin oligomer or antibody can be labeled with peroxidase, alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate. Additives such as 5-amino-2,3-dihydro-1,4-phthalazinedione (also known as LuminolTM) (Sigma Chemical Company, St. Louis, MO) and rate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol) (Sigma Chemical Company, St. Louis, MO) can be used to amplify enzymes such as horseradish peroxidase through a luminescent reaction; and luminogenic or fluorogenic dioxetane derivatives of enzyme substrates can also be used. Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer. In addition, endostatin oligomers o r antibodies may be labeled with colloidal gold for use in immunoelectron microscopy in accordance with methods well known to those skilled in the art.
WO 991b2944 PCT/US99/12278 The proteins and antibodies of the present invention are useful for diagnosing and treating metastatic and angiogenesis-related diseases. One example of metastatic disease is metastatic cancer. Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;
rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, ru.beosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. The Endostatin- oligomers of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Hedobacter pylori).
According to the present invention, endostatin oligomers may be used in combination with other compositions and procedures for the treatment of the above described diseases and conditions. For example, a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with endostatin oligomers and subsequently endostatin oligomers may be administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor.
The endostatin oligomers described above can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art.
These formulations can be administered by standard routes. In 1g general, the combinations may be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route. In addition, the endostatin oiigomers may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the endostatin oligomer is slowly released systemically. Osmotic minipumps may also be used to provide controlled delivery of high concentrations of endostatin oligomers through cannuiae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor. The biodegradable polymers and their use are described, for example, in detail in Brem et al. (1991), which is hereby incorporated by reference in its entirety.
The dosage of an endostatin oligomer of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. For treating humans or animals, between approximately 0.5 mg/kilogram to 500 rng/kilogram of the endostatin oligomer and peptide can be administered. A more preferable range is 1 mg/kilogram to 100 mglkilogram with the most preferable range being from 2 mglkilogram to 50 mg/kilogram. Depending upon the half-life of the endostatin oligomer and peptide in the particular animal or human, it can be administered between several times per day to once a week. It is to be understood that the present invention has application f or both human and veterinary use. The methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
The endostatin oligomer formulations include those suitable for oral, rectal, ophthalmic (including intravitreal or WO 99/62944 PCTlUS99/12278 l xntracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural) administration. The endostatin oligomer formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carriers) or excipient(s): In general, the l0 formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary; shaping the product.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats. and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or mufti-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use: Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
Tn addition to methods of treatment, the present invention also relates to methods of using endostatin oligomers, and active i fragments thereof, and antibodies that bind specifically to endostatin oligomers and their peptides, to diagnose endothelial cell-related diseases and disorders. Diagnosis is accomplished by detection of endostatin oligomers, or antibodies thereto, in body 5 fluids or tissues. Detection may be accompanied by comparison of the detected levels of endostatin oligomers, or antibodies thereto, to normal levels of endostatin oligomers, or antibodies thereto.
Kits for measurement of endostatin oligomers are also contemplated as part of the present invention. Antisera that 10 possess the highest titer and specificity and can detect endostatin oligomers in extracts of plasma, urine, tissues, and in cell culture media are further examined to establish easy to use kits for rapid, reliable, sensitive, and specific measurement and localization of endostatin oligomers. These assay kits include but are not limited 15 to the following techniques; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or 20 dipsticks for rapid monitoring of urine or blood, and immunocytochemistry. For each kit, the range, sensitivity, precision, reliability, specificity and reproducibility of the assay is established. Intra-assay and inter-assay variation is established at 20%, SO% and $0% points on the standard curves of displacement or activity.
The assay kit provides instructions, antiserum, endostatin oligomers and possibly radiolabeled endostatin oiigomers or peptides and/or reagents for precipitation ~ of bound antibody complexes. The kit is useful for the measurement of endostatin oligomers in biological fluids and tissue extracts of animals and humans with and without tumors or angiogenesis-dependent diseases.
Also included in the present invention are kits for the localization of endostatin oligomers or peptides in tissues and cells. This endostatin oligomer immunohistochemistry kit WO 99!62944 PCT/US99/122'78 provides instructions, endostatin oligomer antiserum, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum.
Immunohistochemistry techniques are well known to those skilled in the art. This endostatin oligomer and peptide immunohistochemistry kit permits localization of endostatin oligomers in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical l0 purposes. For example, tumors are biopsied or collected and tissue sections cut with a microtome to examine sites of hyaluronic acid binding link module production. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer.
IS The invention further encompasses a method for identifying and quantitating endostatin oligomer-specific receptors. Labeling of the endostatin oligomers with short lived isotopes enables visualization of receptor binding sites in vivo using positron emission tomography or other modern 20 radiographic techniques. These methods are important for the study of angiogenesis and metastasis in cancer and other angiogenesis-related diseases.
This invention is further illustrated by the following examples, which are not to be construed in any way as imposing 25 limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present 30 invention and/or the scope of the appended claims.
Example 1 Production of Endostatin Monomers and Dimers and Oligomers To produce endostatin monomers, the polymerase chain 35 reaction {PCR) was used to adapt the endostatin cDNA for expression in the pdCs-Fc(D4K) vector, which contains the enterokinase recognition site Asp4-Lys at the junction of the fusion protein. The forward primer was 5'-C AAG CTT CAC
AGC CAC CGC GAC TTC C (SEQ ID N0:3), containing a Hind III site followed by a sequence encoding the N-terminus of endostatin (HSHRDFQPVLHL, SEQ ID N0:4}. The reverse primer was 5'-C CTC GAG CTA CTT GGA GGC AGT CAT G, (SEQ ID N0:5} which was designed to put a translation STOP
codon (anti-codon, CTA) immediately after the C-terminus of endostatin, and this was followed by a Xho 1 site (CTCGAG}.
The PCR product was cloned and sequenced, and the Hind III-Xho 1 fragment encoding endostatin was ligated to the pdCsFc(D4K) vector. Stable clones expressing Fc(D4K)-endostatin were obtained by electroporation of NSIO
cells followed by selection in growth medium containing 100 nM
methotrexate. Protein expression levels were assayed by anti-human Fc ELISA as described in Lo, K. M. et al. ( 1998) Protein Engineering 11:495-500 and confirmed by SDS-PAGE.
The best producing clone was sub-cloned by limiting dilutions.
The Fc-human endostatin fusion protein was purified on Protein A sepharose using 100mM citric acid, pH=3.0 as elution buffer. Enterokinase and trypsin digestions were carried out resulting in two forms of cleaved endostatin. For trypsin, the ratio of enzyme to protein (w/w) was 1:200, 18 hours room temperature resulting in an endostatin molecule with four amino acids, HSHR, cleaved from N-terminus. Enterokinase digestion led to a product with one additional amino acid, leucine, at the N-terminus of endostatin. Both cleaved products were purified further using heparin Sepharose and SP sepharose (Pharmacia, Bridgewater, New Jersey).
Disulfide-bonded human endostatin dimer was produced by replacing the restriction fragment encoding the N-terminal sequence of human endostatin in pdCs-Fc(D4K)-hu Endo with a double-stranded oligonucleotide duplex encoding the same sequence but with the Q7 codon changed to C to generate pd WO 99/62944 PCTlUS99/12278 ' 23 Cs-Fc(D4K)-hu Endo (C7): This plasmid was transfected into myeloma cells and the Fc-endostatin dimer was purified from conditioned media utilizing protein A affinity chromatography as described in Lo, K.M. et al.; (1998) Protein Engineering 11:495-500. After the Fc-endostatin dimer was cleaved with enterokinase the endostatin dimer resolved from the Fc portion by gel filtration over S-200.
The endostatin-NCI trimer was produced as follows. The NC I protein was produced using two primers, 5' GAT CGG CCC
1o AGC CGG CCC ATC ATC ACC ATC ACC ATG ACG ATG
ACG ATA AGT CAG GGC AGG TGA GGC TCG CTA CAC
GCA GG 3' (SEQ ID NO:6) and 5'GAT CGG ATC CCT ACT
TGG AGG CAG TCA TGA AGG 3' (SEQ ID N0:7). These primers were used to amplify from the SGQVRLWATRQ amino acid sequence (SEQ ID N0:8} through the C terminus of human collagen I8 using PCR. The PCR products were cloned in the Sfil-BamHl site in pSecTag2a (Invitrogen, San Diego, California}.
Stable 293T cell transfectants were selected in zeocin and conditioned media eluted from Ni-agarose (QIAGEN, Valencia, California) with 250 mM imidazole/PBS followed by dialysis into PBS.
Example 2 Zinc-Dependent Dimers Observed in Crystals of Human Endostatin The X-ray structure was determined from crystals obtained at 4° C by hanging drop vapour diffusion, with equal volumes of 10 mg/ml protein in 20 mM Tris-HCl pH 8.5, 150 mM NaCI and 50 mM Tris-HCI pH 8.5, 6% PEG 8K, 150 mM NaCI and 2 mM
MgC 1 mixed at room temperature in the drops, equilibrated against 0.5 ml of the later buffer. Crystals (C2 a=92.76 t~, b=74.27 ~, c=137.80 ~, 13=102.56 have 4 monomers in the asymmetric unit. Crystals were transferred to 15% PEG 6K, 20%
glycerol, 50 mM Tris-HC 1 pH 8.5 and 150 mM NaCI for about 20 seconds before flash cooling in a stream of cold nitrogen. Data were collected at the CHESS Al station (~,=0.92A) with the 80 mm 2K CCD (bined mode) (0.5 degree oscillations). Data were integrated and scaled with DENZO and SCALEPACK (HKL
Research, Charlottesville, Virginia). Most of the subsequent processing used the CCP4 programs (T. A. Jones ( 1992) "Molecular Replacement" CCP4, 92-105; G. J. Kleywegt and T. A. Jones { 1994) "From First Map to Final Nlodel " CCP4, 59-66).
Following the above methods it was determined that the zinc binding site of human endostatin is tetrahedral with three zinc Iigands from the N-terminal loop, histidines 1, 3, and 11, and a fourth ligand, aspartic acid 76, from the loop between the E and F 13-strands (Fig. 1 and Fig. 2). The zinc binding site most closely resembles that from zinc proteases. Atomic absorption spectroscopy confirmed that zinc is a constituent of the Fe-endostatin chimeras described above of both human and murine endostatin in solution as shown below in Table I.
Table I
Protein N-terminusEndostatin Zinc Zinc per Endostatin sequence ConcentrationConcentrationMonomer (mg/mL) (micromolar) hE-Ek LHSHRD 4 176 0.9 hE-Tryp D 3.8 14 0.1 hFc-hE 0.4 18 0.9 2 79 0.9 The first line of Table I also demonstrates that enterokinase treated Fc-human endostatin; which yields full length endostatin, also showed approximately one atom of zinc per endostatin monomer. Trypsin cleaved Fc-human endostatin, which results in Ioss of the first four residues of endostatin, contains no detectable zinc (second line of Table I). These results are consistent with the human endostatin crystal structure that shows histidines at residues I and 3 to be two of the zinc ligands.
A number of two-fold symmetric dimers are evident in the packing of the four endostatin monomers in the asymmetric unit 5 of the human endostatin crystals. One dimer interface is formed by the contact of two solvent exposed phenylalanines on the a I cc helix from each monomer, a prominent non-polar patch suggested as a potential inter-domain interaction site in murine endostatin (residues F31 and F34 in Fig. 3). Another dimeric contact, that 10 would be zinc dependent, is formed between the projecting N-terminal loops of two monomers; each loop ordered around a zinc ion (Center in Fig. 3a). This contact is formed primarily by three residues that project from the rim of the N-terminal loop:
arginine 4, phenylalanine 6, and glutamine 7 (Fig. 3b). The 15 glutamines at residue 7 of each monomer contact each other forming a hydrogen bond at the center of the interface. Each ring of the phenylalanines at residue 7 contacts a non-polar patch containing Ieucine 78 and valine 72 of the adjacent monomer.
Arginine 4 from each monomer forms hydrogen bonds with two 20 main chain carbonyl oxygens on the loop containing the aspartic acid zinc ligand at residue 76 (carbonyls at residue 75 and 76) (Fig. 3b). This dimeric interaction could only occur if the N-terminal loops were ordered as the result of binding zinc, and in turn, the zinc binding would probably be stabilized by the 25 dimer interface.
Example 3 Zinc Binding of Endostatin is Essential for Anti-Angiogenic Activity As described in Example 2, it was determined that histidines l, 3, 11 and aspartic acid 76 coordinate the zinc ion.
Based upon this discovery, point mutations were introduced into the endostatin protein to change the Zn2+ ligands to alanines. One double (H1/3A) and two single (Hl I A and D76A) mutants were 3S created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California) in an E. coli expression vector.
The mutations were confirmed by sequence analysis.
Wild-type endostatin and the three mutants were purified in parallel and tested in the Lewis lung carcinoma model as previously described in 0'Reilly, M. S. et al., Cell 88:277 (1997) and T. Boehm et al. Nature 390:404 (1997). The different endostatin preparations were compared on a sodium dodecyl sulfate (SDS) gel under reducing and non-reducing conditions and no significant difference in the purity and cross-linking pattern IO was observed. SDS-PAGE of endostatin purified from E. coli showed a protein ladder including monomers, dimers, trimers and higher molecular weight complexes caused by cross-linking of endostatin to other endostatin molecules via disulfide bond formation.
Unlike the wild-type endostatin, none of the three mutants of endostatin regressed Lewis lung carcinoma (data not shown).
Although the . Hl/3A endostatin mutant was weakly active, the weak activity might be due to either a distorted conformation caused by using two histidines from the His.-tag to coordinate the Zn 2+ ion, or by the loss of the ability to bind zinc.
Example 4 Scatter of Pre formed Endothelial Structures by Endostatin Dimers In order to determine the ability of endostatin monomers and dimers to influence morphogenesis, a matrigel tube-formation assay was used. The assay utilizes endothelial cells plated on extracellular matrix proteins that spontaneously aggregate and assemble into densely rnulti-cellular capillary-like tubular structures. The assay was performed as follows.
HUVEC (human umbilical vein endothelial cells), passage <10, isolated from single donor (Clonetics, San Diego, California), were cultured in EBM media (Clonetics, San Diego, California) supplemented with EGM-2-MV growth factors including 5% FCS, hVEGF, hFGF, ( 10 ng/ml), hydrocortisone ( 1 WO 99/62944 PCT/US99/t2278 ~Cg/ml), and GA-1000 (0.1%) at 37° C, 5% CO2, Cells were plated in 1 ml at 40,000 cells/well into 24 well plates containing complete media as above. These wells had been pre-coated at 4° C
with matrigel, a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse -sarcoma (Becton DickinsonJCollaborative, Franklin Lakes, New Jersey) at 300 ,ul/well. Human endostatin derivatives in PBS
( 10-2000 nM) were added either at the time of addition of the cells to the matrigei-coated plates, or after 16 hours of tube formation. Recombinant hHGF (R&D Systems, Minneapolis, Minnesota) was added at 50 ng/ml.
For inhibition of endostatin oligomers by endostatin monomers, pre-formed tubes were pre-incubated with endostatin monomer (2000 nM) for 30 minutes prior to oligomer treatment.
Wells were subsequently incubated at 37° C, 5% CO2, and then photographed under phase contrast microscopy at 40-200x.
Identical results and procedures have been obtained with human microvascular, pulmonary and umbilical artery primary endothelial cultures.
An antibody Fc-endostatin fusion protein containing endostatin at the C-terminus potently inhibited tube formation when included at the time of plating with cells actually becoming more dispersed over time rather than coalescing into tubular structures (data not shown). Identical results were observed with primary cultures of human microvascular, umbilical vein, umbilical artery and pulmonary artery endothelial cells (data not shown). The rapidity of this effect argued against a primary effect on cell growth, and cell counts, BrdU staining and 3H
incorporation did not reveal significant stimulation of proliferation versus untreated cells during this time interval (data not shown).
Intriguingly, after enterokinase cleavage of the Fc-endostatin fusion into Fc and endostatin, neither Fc nor endostatin singly, nor the combination of Fc and endostatin demonstrated inhibitory activity (Figure 4). Recombinant endostatin from baculovirus and yeast un-fused to Fc were also ineffective (data not shown). However, an artificially dimerized endostatin created by utilizing a close contact between adjacent glutamine-7 residues predicted by the dimeric crystal structure of endostatin, was found to exhibit scatter activity. Mutation of endostatin giutamine-7 to cysteine and fusion to antibody Fc allowed formation of a novel intermolecular cysteine bond at cysteine-7, with enterokinase cleavage liberating the free endostatin dimer migrating at 40 kDa and 20 kDa under non-reducing and reducing conditions respectively (data not shown). Both mouse and human endostatin dimers potently reproduced the inhibitory activity of the Fc-endostatin fusion protein on tube formation with ICso of approximately 10 nM (Figure 4) and were inactive against bovine capillary endothelial cell or HUVEC proliferation (data not shown).
During endostatin dimer-induced scattering, rapidly demarginating cells appeared as early as 1-2 hours with prominent lamellipod and pseudopod formation (data not shown).
Confocal microscopy with phalloidin-FITC revealed a dramatic reorganization of the actin cytoskeleton within two hours with prominent stress fiber formation (data not shown), consistent with a signaling pathway linked to rapid actin polymerization. As visualized by electron microscopy, endostatin oligomer-induced scattering was accompanied by destruction of tubular lumens and by cellular elongation (data not shown).
The scatter factor activity of the endostatin oligomers described herein is specific. Although collagen XV (c 15) and collagen XVIII, from which endostatin monomers are derived, exhibit 60% amino acid identity in the endostatin domain, and share the NC 1 trimerization domain, collagen XV oligomers did not act as scatter factors (Figure 4). Fc fusion protein containing the c I5 endostatin-Iike domain was unable to scatter tubules, in contrast to Fc endostatin dimers, and also could not antagonize the effects of Fc endostatin dimers (data not shown). Fc-angiostatin or monomeric angiostatins also did not inhibit tube formation, WO 99162944 PCT/US99/122'18 consistent with the complete lack of structural homology between endostatin and angiostatin (data not shown). Overall, only oligomeric endostatin compositions displayed scatter activity.
Additionally, pretreatment of established tubules with excess of endostatin monomer ( 1000 nM) strongly blocked the ability of endostatin dimers (20-50 nM) to scatter cells (data not shown). This is potentially consistent with endostatin monomers acting in a dominant negative fashion to block endostatin oligomers, for example, by non-productively occupying a receptor that requires oligomer induced cross-linking for activity.
Example 5 Scatter of Pre formed Endothelial Structures by Endostatin-NCI
Trimers Human and marine NC1 proteins were isolated from transfected 293T cell supernatants as described above in Example 1. Trimerization was confirmed by cross-linking the trimerized complexes and resolving the products by non-reduced denaturing polyacrylamide gel electrophoresis (PAGE). Cross-linking was carried out in the presence of 5mM Ethylene Glycol-bis(Succinimidyisuccinate) (EGS), at room temperature for 20 minutes. The EGS was dissolved in DMSO and was diluted ten fold in PBS. The reaction was stopped by adding Tris at a final concentration of 100mM. The resulting gel produced an approximately 120 kDa species upon EGS cross--linking (data not shown).
The marine and human NCl-endostatin trimers identically inhibited endothelial tube formation with an ICSO of approximately 10 nM (Figure 4), again consistent with dependence of activity on endostatin domain oligomerization. The NCI-endostatin trimer was slightly less effective than the endostatin dimers at higher concentrations, possibly reflecting decreased potency from non-covalent versus covalent oligomerization.
Scattering results obtained with the endostatin/NC 1 trimer were similar to the endostatin dimer. The endostatin/NC 1 trimer induced scattering, rapidly demarginating cells appeared as early as 1-2 hours with prominent lamellipod and pseudopod formation 5 (data not shown). Confocal microscopy with phalloidin-FITC
revealed a dramatic reorganization of the actin cytoskeleton within two hours with pronunent stress fiber formation, consistent with a signaling pathway linked to rapid actin polymerization. As visualized by electron microscopy, endostatin 10 oligomer-induced scattering was accompanied by destruction of tubular lumens and by cellular elongation (data not shown).
Pretreatment of established tubules with excess of endostatin monomer (1000 nM} strongly blocked the ability of the NCI-endostatin trimer {20-50 nM) to scatter cells (data not 15 shown). As stated above, this is potentially consistent with endostatin monomers acting in a dominant negative fashion to block endostatin oligomers; for example, by non-productively occupying a receptor that requires oligomer induced cross-linking for activity.
Example 6 Endostati~c Oligomers Comprise a New Ciass of Scatter Factors The oligomeric endostatin-induced cell scattering and cytoskeletal reorganization were reminiscent of morphogenic changes evoked by Hepatocyte Growth Factor/Scatter Factor (HGF/SF), which with the related Macrophage Stimulatory Protein (MSP) comprises a scatter factor family regulating cell motility, dispersion, proliferation and apoptosis. Although Human Umbilical Vein Endothelial Cells (HUVECs) abundantly express the HGF receptor c-met 16, in contrast to endostatin oligomers, HGF was completely unable to scatter HUVEC tubules (data not shown}. Conversely, while HGF potently dis-aggregated and scattered MDCK and HepG2 cell aggregates cultured on plastic, oligomeric endostatin derivatives were inactive (data not shown).
MSP was also unable to elicit HUVEC scatter (data not shown).
WO 99/62944 PCT/US99/t2278 Notably, it is well established that HGF actually promotes tube formation in collagen matrices as opposed to the anti-tubulogenic effects of endostatin described here. Endostatin oligomers therefore define a novel scatter factor class, both structurally and functionally distinct from: the previously unique HGF/MSP
family.
Modifications and variations of the present method will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.
References Brem, H. et al. (1991). Interstitial chemotherapy with drug polymer implants for the treatment of recurrent gliomas. J:
Neurosurg. 74, 441-46.
Chen, C., Parangi, S., Tolentino, M. J., and Folkman; J. (1995).
A strategy to discover circulating angiogenesis inhibitors generated by human tumors: Cancer Res. 55, 4230-4233.
Coleman, J. E. (1992). Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu. Rev.
Biochem. 61, 897-946.
Cunningham, B. C., Bass, S., Fuh, G., and Wells, J. A. ( 1990).
Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science 250, 1709-1712.
Cunningham, B. C., Mulkerrin, M. G., and Wells, J. A. ( 199I ).
Dimerization of human growth hormone by zinc. Science 253, 545-548.
Folkman, J. ( 1989}. What is .the evidence that tumors are angiogenesis dependent?. J. Natl. Cancer Inst. 82, 4-6.
Good, D. J., Polverini, P. J., Rastinejad, F., Le Beau, M. M., Lemons, R. S., Frazier, W.A., and Bouck, N. P. (1990). A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Nat. Acad. Sci. USA. 87, 6624-6628.
Hanahan, D., Folkman, J. (i996}. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364.
Hohenester, E, Sasaki, T., Olsen, B. R:, and Timpl, R. ( 1998}.
Crystal structure of the angiogenesis inhibitor endostatin at 1.5 ~
resolution. The EMB4 J. 17; 1656-1664.
Hori, A., Sasada, R., Matsutani, E., Naito, K., Sakura, Y., Fujita, T., and Kozai, Y. ( 1991 ). Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor. Cancer Res. 51, 6180-6184.
Kandel, J., Bossy-Wetzel, E., Radvany, F., Klagsburn, M., Folkman, J., and Hanahan, D. ( 1991 ). Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. Cell 66, 1095-1104.
Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and Ferrara, N. (1993). Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362, 841-844.
WO 99162944 PCT/US99/1227$
Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., and Ullrich, A. (1994). Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 367, 576-579.
Murga.ki, Y. et al. (1995). Mouse Co118a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones. Proc. Natl. Acad. Sci. USA 92, 8763-8767.
O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R.
A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. ( 1994). Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79, 315-328.
O'Reilly, M. S., Holmgren, L., Chen, C. and Foikman J, (1996).
Angiostatin induces and sustains dormancy of human primary tumors in mice. Nature Med. 2, 689-692.
O'Reilly, M. S. et al. { 1997): Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285.
Oh, S. P. et al. (1994). Isolation and sequencing of cDNAs for proteins with multiple domains of Gly-Xaa-Yaa repeats identify a distinct family of collagenou proteins.
Parangi, S., O'Reilly, M., Christofori, G., Holmgren, L., Grosfeld, J., Folkman, J., and Hanahan, D: ( 1996).
Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc. Natl. Acad. Sci. USA 93, 2002-2007.
Rastinejad, F., Polverini, P. J., and Bouck, N. P. ( 1989).
Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56, 345-355.
a Rehn, M., Pihlajaniemi, T. (1994}. Alphal(XVIII), a collagen chain with frequent interruptions in the collagenous sequence, a distinct tissue distribution, and homology with type XV collagen.
Proc. Natl. Acad. Sci. USA 91, 4234-4238.
Vallee, B. L. and Auld, D. S. ( 1990). Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647-5659.
to Zetter, B.C. (1998). Angiogenesis and Tumor Metastasis. Annu.
Rev. Med. 49, 387-424.
ENDOSTATIN PROTEIN AND METHODS
OF USING THE SAME
Cross Reference to Related Applications This application claims priority to provisional application Serial No. 60/087,890 filed June 3, 1998; provisional application Serial No. 60J092,393 filed July 10, 1998; and provisional application Serial No. 60/098,790 filed September l, 1998. Each of the above-referenced applications is incorporated herein in its entirety.
Field of the Invention The present invention relates to the fields of oncology, angiogenesis and morphogenesis and more particularly to novel protein oligomers comprising endostatin protein. The endostatin oligomers are useful fox inhibiting endothelial cell tubule formation, regulating cellular morphogenesis, and treating metastatic cancers and angiogenesis-dependent diseases.
Background of the Invention Several lines of direct evidence now suggest that the growth and persistence of solid tumors as well as their metastases to distant organs is critically dependent upon angiogenesis, or the recruitment of new blood vessels (Folkman, 1989; Hori et al., WO 99/b2944 PCT/US99/12278 1991; Kim et al., 1993; Millauer et al., 1994). Angiogenesis not only provides the increased nutrients and pathways for the removal of waste needed for the expansion of the tumor, but it also facilitates tumor metastasis by providing a route for tumor cells to leave the primary site and enter the bloodstream (letter, 1998). In particular, angiogenesis increases the entry of tumor cells into the bloodstream by providing an increased density of immature, highly permeable blood vessels that have thinner basement membranes and fewer intracellular junction complexes than normal mature vessels (letter, 1998).
It is postulated that the angiogenic phenotype is the result of a net balance between both positive and negative regulators of neovascularization (Good et al., 1990; O'Reilly et al., 1994;
Parangi et al., 1996; Rastinejad et aL, 1989). Tumors themselves, along with other accessory host cells such as macrophages, mast cells and lymphocytes; stimulate angiogenesis by up-regulating their production of a variety of angiogenic factors, including the fibroblast growth factor (FGF). (Kandel et al., 1991 ) and vascular endothelial cell growth factor/vascular permeability factor (VEGF/VPF) (letter, 1998). However, many malignant tumors also generate inhibitors of angiogenesis, including angiostatin protein and thrombospondin (Chen et al., 1995; Good et al., 1990; O'Reilly et al., 1994; U.S. Patent No. 5,639,725).
Several angiogenic and anti-angiogenic proteins are stored as inactive precursors in the blood or basement membranes (Hanahan, 1996). One example of such an inactive precursor is endostatin protein. Endostatin protein is an approximately 20 kDa C-terminal globular domain of the collagen-like protein, collagen XVIII (c18) which is localized in the basement membrane around blood vessels {Oh et al., 1994). Endostatin is stored in vivo as the C-terminal portion of the c 18 protein (Rehn et al., 1994;
Muragaki et al., 1995) and fragments of c 18 longer than endostatin are believed not to inhibit endothelial cell proliferation (O'Reilly et al., 1997). Endostatin was first isolated from a hemangioendothelioma cell line for its ability to inhibit the WO 99!62944 PCT/US99I12278 ' 3 proliferation of capillary endothelial cells as described in U.S.
Patent Number 5,854,205, which is hereby incorporated by reference.
Although it is known that endostatin is stored as an inactive precursor, it is not known how endostatin is activated. Previous x-ray crystallographic studies indicated that endostatin does not contain the characteristic Ca2+ binding sites that are active in selectins, a similar group of molecules {Hohenester et al., 1998).
These same studies indicated that no metal ions were associated with human endostatin.
Metal ions are often involved in the biologic activity of proteins. In particular, the zinc cation is a critical component of many proteins and plays a key role in a host of biological processes (Coleman, 1992; O'Reilly et al, 1996, Vallee et al.
1990). In most cases, zinc is directly involved in the catalytic activity, however structural roles for zinc have also been described. For example, zinc is involved in the dimerization of human growth hormone and increases the affinity of human growth hormone for the prolactin receptor by approximately 8,000 fold {Cunningham et al., 1990; Cunningham et al., 1991 ).
Summary of the Lnvention The present invention includes protein oligomers comprising more than one endostatin protein monomers, wherein the oligomer has scatter factor activity. Endostatin protein is a carboxy-terminal region fragment of collagen XVIII having a molecular weight of approximately 20 kDa as determined by reducing gel electrophoresis and I8 kDa as determined by non-reducing gel electrophoresis. In one embodiment of the present invention, the protein oligomer comprises a dirner of endostatin monomers. In an alternate embodiment, the protein oligomer comprises more than one NCI region fragment of collagen XVIII
having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC 1 fragment contains an endostatin monomer. In a preferred embodiment, the protein oligomer comprising more than one NC 1 region fragment is a trimer. The protein oligomers of the present invention may optionally comprise a metal component, which is preferably zinc.
The novel protein oligomers described herein constitute a novel class of scatter factors. The protein oligomers of the present invention have anti-tubulogenic effects and induce reorganization of the actin cytoskeleton, destruction of tubular lumens, and elongation of cells. The novel protein oligomers axe also anti-tumorigenic and anti-angiogenic.
Also included in the present invention are methods of using the protein oligomers comprising endostatin protein. The protein oligomers described herein may be used to inhibit tubulogenesis and tumorigenesis.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
Brief Description of the Figures Figure 1. Structure of Hurrcan Endostatin 13 strands are labeled in sequential order A-P, a helices are denoted, and zinc is shown as a sphere.
Figure 2. The N-terminal Loop and Zinc Binding Site of Human Endostatin The zinc (black circle) ligands histidines 1, 3, and 1 i and aspartic acid 76, as well as the second shell of interactions that position the zinc ligands, glutamic acid 175, residue 11 carbonyl oxygen, and arginine 4 from the N-terminal loop of an adjacent molecule in a dimer are shown as ball and stick models ' 5 Figure 3A-B. A Zinc Dependent Dimer in Human Endostatin Crystals Figure 3A. The zinc (black sphere) site N-terminal loops of two monomers contact across a central dyad axis. Glutamine 7, phenylalanine 6, and arginine 5 of the loop project from one monomer to the next. Also shown are two phenylalanine rings, residues 31 and 34, that project from an endostatin oc helix and form another dimeric contact in the crystal.
Figure 3B. Contacts in the interface of the dimer seen in IO crystals of human endostatin. Zinc (black sphere) ligands have open bonds, interface residues have solid bonds. The path of the polypeptide chains of the two monomers are shown as tubes. The solvent accessible surface buried in this dimer interface is 403 ~2 (probe size = 1.4 ~.) per monomer.
Figure 4. Oligomeric Endostatins Exhibit Scatter Factor Activity HUVEC tubules on matrigel were pre-formed for 16 hours and treated with human endostatin monomer, human Fc endostatin dimer, human Fc dimers, human Fc-endostatin(C7) dimers, human endostatin(C7) dimers, human endostatin/NC1 trimers, human Fc collagen 15 dimers, or Fc angiostatin dimers.
Oniy endostatin dimers and trimers exhibited scatter activity and inhibited tubule formation.
Detailed Description The present invention provides a new class of scatter factors comprising endostatin oligomers. Oligomers in this new class of scatter factors act as "anti-matrix" scatter factors and are capable of inhibiting endothelial tubule assembly evoked by the presence of extracellular matrix proteins. The endostatin oligomers are additionally anti-angiogenic and anti-tumorigenic.
Although the endostatin oligomers have been defined functionally, it is to be understood that this functional definition in no way limits the bioactivity of the endostatin oligomers.
WO 99/62944 PCTfUS99/I2278 The protein oligomers of _one embodiment of the present invention comprise a plurality of endostatin monomers, wherein the endostatin monomers are approximately 20 kDa proteins as determined under reduced gel electrophoresis or approximately 18 kDa as determined under non-reduced gel electrophoresis, and are characterized by their ability to inhibit proliferating cultured endothelial cells. The endostatin oligomers are carboxy-region fragments of collagen-like molecules such as collagen XVIII, and may be derived from any mammal. In a preferred embodiment, the endostatin monomer begins at approximately amino acid position 1132 of murine collagen XVIII, and correlates to the human endostatin fragment of SEQ ID NO:1 shown below.
HSHRDFQPVLHLVALNSFLSGGMRGIRGADFQCFQQARAVG
LAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSW
EALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKS V WHGSD
PNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASC
HHAYIVLCIENSFMTAS
As described below, amino acid substitutions may occur in the sequence of endostatin which still yield a functional endostatin protein. For example, when the above gene sequence is recombinantly expressed, an observable doublet of protein results, both versions of which are functional endostatin proteins.
In addition to the above endostatin protein, the following endostatin variant occurs, which is the former protein minus the first four amino acids: This demonstrates the variability of functional endostatin protein molecules. Therefore, in an alternate embodiment, the endostatin monomer correlates to the human endostatin fragment of SEQ ID N0:2 shown below.
DFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAG
TFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEAL
FSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNG
RRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA
YIVLCIENSFMTAS
The terms "endostatin" and "endostatin monomer" are synonymous and include naturally occurring, recombinant, or synthetic endostatin proteins that contain conservative, or "silent"
amino acid substitutions, deletions and additions, yet upon oligomerization retain scatter factor activity. The term "scatter factor activity" refers to the disruption of endothelial tubule formation as determined by a matrigel tube-formation assay. In a preferred embodiment, the endostatin monomers are modified at the seventh amino acid such that glutamine is replaced with cysteine. Replacement of glutamine with cysteine facilitates dimerization or oligomerization between endostatin monomers.
The term "endostatin monomer" also includes shortened proteins wherein one or more amino acid is removed from either or both ends of an endostatin monomer, or from an internal region of the protein, yet upon oligomerization retain scatter factor activity.
The term "endostatin monomer" also includes lengthened proteins or peptides wherein one or more amino acids is added to either or both ends of an endostatin monomer, or to an internal location, yet upon oligomerization retain scatter factor activity. One example of such a modification is the addition of tyrosine to the first position. Tyrosine labeled molecules may be further labeled with lasiodine for use in assays. Labeling with other radioisotopes or chemicals such as ricin may also be useful in providing a molecular tool for destroying the target cells containing endostatin oligomer receptors. Endostatin monomers can also be recombinantly fused to other proteins or peptides, such as a Fc portion of an antibody as described in the Examples below.
Additionally, silent substitutions of amino acids, are well known in the art and are intended to fall within the scope of the appended claims. Silent substitutions occur when the replacement of an amino acid with a structurally or chemically similar amino acid does not significantly alter the structure, conformation or , 8 activity of the protein. Also included in the definition of the term "endostatin monomer" are modifications of the protein, its subunits and peptide fragments. Such modifications include substitutions of naturally occurring amino acids at specific sites with other molecules, including but not limited to naturally and -non-naturally occurring amino acids. Such substitutions may modify the bioactivity of endostatin oligomers, such as by increasing or decreasing the scatter factor activity, and produce biological or pharmacological agonists or antagonists.
l0 In one embodiment, the protein oligomers are endostatin dimers. The present invention includes a novel class of scatter factors that includes endostatin dimers. The endostatin dimers of the present invention progressively disperse established tubes into constituent cells as early as 2-3 hours and without obvious toxicity. During the endostatin dimer induced scatter, dramatic reorganization of the cytoskeleton can be observed along with destruction of tubular lumens and cellular elongation. The endostatin oligomers differ from the previously described Hepatocyte Growth Factor/ Scatter Factor (HGF/SF) class of 2o scatter factors. In contrast to the HGF/SF class of scatter factors that promote tube formation, the endostatin oligomers of the present invention have anti-tubulogenic effects. The endostatin oligomers also affect different types of cells as compared to the HGF/SF class of scatter factors. In addition to scatter activity, the endostatin dimers of the present invention are also capable of anti-tumorigenic and anti-angiogenic activity when bound to a metal such as zinc.
In an alternate embodiment, the invention provides protein oligomers comprising a plurality of endostatin/NCl proteins. The endostatin/NC 1 proteins of the present invention are approximately 38 kDa C-terminal region fragments of collagen XVIII that each contain the approximately 20 kDa endostatin proteins described above. The present invention describes for the first time that endostatin/NC 1 trimers are included in the new class of scatter factors described above. Endostatin/NC 1 trimers induce scattering of endothelial cell tubular structures as well as other cellular morphogenic changes in a manner similar to endostatin dimers. The anti-tubulogenic activity of the endostatin/NC 1 trimers can also be inhibited by endostatin monomers.
In another embodiment, the protein oligomers described above comprise Pndostatin monomers that are fusion proteins.
The endostatin fusion proteins may comprise endostatin and anti-angiogenic molecules, angiogenic molecules, and/or molecules that facilitate dimerization of the endostatin monomers. In a preferred embodiment, the endostatin fusion proteins comprise endostatin and the Fc portion of an antibody; wherein the Fc portion of the antibody promotes dimerization. The Fc portion may be derived from the IgG, IgE, IgA or IgM isotype, however, the preferred isotype is IgG.
In a further embodiment, the protein oligomers, dimers and monomers are bound to a metal ion, preferably a zinc ion.
The, invention provides for the first time that endostatin contains a zinc binding site and requires metal binding for activity. While prior attempts to study crystallized endostatin resulted in disordered residues and inaccurate results, the inventors have expressed endostatin monomers in such a manner that the residues are not disordered and have identified a zinc binding domain on endostatin. In the present invention, human endostatin has been expressed as a secreted protein in a murine myeloma cell line as a chimera with the Fc domain of IgG-1 and released from the Fc portion by enterokinase digestion (as more specifically described below). Additionally human endostatin crystals were grown at a higher pH where the N-terminal histidines would not be charged.
These modifications of prior art methods have allowed the inventors to accurately identify endostatin as a zinc binding protein.
Using the above methods, the inventors discovered that the zinc binding site of human endostatin is tetrahedral with three zinc ligands from the N-terminal loop, histidines 1, 3, and l l, and WO 99162944 PCTlUS99/12278 ' 10 a fourth ligand, aspartic acid 76, from the loop between the E and F 13-strands (Fig. 1 and Fig. 2). The inventors also describe for the first time zinc dependent dimeric contacts between endostatin monomers in human endostatin crystals. Based upon these findings, the present invention provides novel endostatin compositions bound to zinc wherein zinc is essential to the anti-tumorigenic activity of the endostatin compositions, and methods of using these compositions. In a preferred embodiment, the Fc-endostatin dimers are bound to zinc. In another preferred embodiment, NC 1/endostatin trimers are bound to zinc.
The present invention also encompasses methods of activating endostatin in vitro or in vivo comprising the binding of one or more metal ions to endostatin oligomers. In a preferred embodiment, the metal ion is zinc, and in a further preferred embodiment, one zinc molecule is bound per endostatin monomer. Activation of endostatin includes increased anti-angiogenic activity, increased tumorigenic activity and/or increased scatter factor activity. In one embodiment, zinc binding causes dimerization of endostatin monomers and consequently activates endostatin. Also included in the invention are methods of forming endostatin oligomers by the addition of a metal ion, preferably a zinc ion.
Typically, the isolated, endostatin oligomers of the invention are at least about 80% pure, usually at least about 90%, and preferably at Ieast about 95 % as measured by band intensity on a silver stained gel. The phrases "isolated", "biologically pure"
or "substantially pure" refer to material which is essentially free from at least some of the components which normally accompany it as found in its native state. Other important terms that are used herein are defined as follows. The terms "a", "an" and "the" as used herein are defined to mean one or more and include the plural unless the context is inappropriate. "Anti-tumor activity"
and "anti-tumorigenic" refer to the ability of an endostatin oligomer to regress a tumor. "Regression" refers to the reduction of tumor mass or size. The term "anti-angiogenic" refers to inhibition of vascularization. Additionally, "substantial sequence homology" means at least approximately 70% homology between a known amino acid residue sequence in an endostatin oligomer, preferably at least approximately 80% homology, more preferably at least approximately 90% homology. Homology can be determined by sequence identity, or by using a well-known computer program, such as DNA Star or GeneJockey, on default setting parameters.
The endostatin and NC 1 monomers that constitute portions of the protein oligomers described above may be isolated from body fluids and tissues including, but not limited to, serum, urine and ascites fluid. Endostatin monomers and NC 1 may also be produced from recombinant sources, genetically altered cells implanted into animals, tumors, cell cultures and other sources.
Recombinant techniques include gene amplification from DNA
sources using amplification techniques such as the polymerase chain reaction (PCR), and gene amplification from RNA sources using amplification techniques such as reverse transcriptase/PCR.
The endostatin monomers may be produced by polypeptide synthesis, or derived by ~n vitro enzymatic catalysis of larger, encompassing polypeptides such as collagen XVIII to yield active endostatin monomers. In a preferred embodiment, the endostatin monomers of the present invention are produced from a recombinant expression system, wherein the cell is Pichia pastoriS.
The endostatin oligomers of the present invention have several uses. The endostatin oligomers are particularly useful for inhibiting endothelial cell tubule formation and for the study of morphogenesis. Endostatin oligomers, and active fragments thereof, are also useful for treating metastatic cancers and tumors as well as angiogenesis-related cancers and diseases.
For example, the endostatin oligomers may be employed to treat metastatic and angiogenesis-dependent cancers and other angiogenesis-related diseases. One example of metastatic disease is metastatic cancer. Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, fox example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; and wound granulation. The endostatin oligomers of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids.
They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helobacter pylori}.
The endostatin oligomers may also be used to develop affinity columns for isolating antibodies directed toward the endostatin oligomers. Those antibodies may be isolated and purified, followed by amino acid sequencing. Also, polypeptides that bind to endostatin oligomers with high specificity and avidity may be labeled with a label or reporter group and employed for visualization and quantitation in the assays described herein using detection techniques such as autoradiographic and membrane binding techniques. The reporter group or label is commonly a fluorescent or radioactive group or an enzyme. Such applications provide important diagnostic and research tools.
Endostatin oligomers can also be employed to develop affinity columns for isolation of endostatin oligomer receptors.
Isolation and purification of such receptors may be followed by amino acid sequencing. Using this information, the gene or genes coding for the receptors can be identified and isolated. Next, cloned nucleic acid sequences may be developed fox insertion into vectors capable of expressing the receptors.
Applicants' invention also encompasses recombinant expression systems wherein nucleic acids encoding the protein constituents of the endostatin oligomers described herein are contained in a single cell. The nucleic acids encoding the oligomeric constituents may be in a single vector or multiple vectors. In a preferred embodiment, the recombinant expression system is Pichia pastoris. The biologically active endostatin oligomers and nucleic acid sequences corresponding to the proteins are useful for modulating endothelial processes in vivo, and for diagnosing and treating endothelial cell-related diseases, for example by gene therapy.
Nucleic acid sequences that correspond to, and code for, the protein constituents of endostatin oligomers can be prepared based upon the knowledge of the amino acid sequence, and the art recognized correspondence between codons (sequences of three nucleic acid bases), and amino acids. Because of the degeneracy of the genetic code, wherein the third base in a codon may vary, yet still code for the same amino acid, many different possible coding nucleic acid sequences are derivable for any particular protein or peptide fragment. Alternatively, the nucleic acid sequence may be derived from a gene bank using oligonucleotides probes designed based on the N-terminal amino acid sequence and well known techniques for cloning genetic material. Nucleic acid sequences are synthesized using automated systems well known in the art.
Either the entire sequence may be synthesized or a series of smaller oligonucleotides are made and subsequently ligated together to yield the full length sequence. The genes encoding constituents of endostatin oligorners may also be isolated from cells or tissue that express high levels of endostatin monomers or NC 1 by ( 1 ) isolating messenger RNA from the tissue, (2) using reverse transcriptase to generate the corresponding DNA
sequence and then (3) using PCR with the appropriate primers to amplify the DNA sequence coding for the active sequence.
w0 99/62944 PCT/US99/12278 ' 14 The isolated, recombinant or synthetic endostatin oligomers described herein are useful for generating antibodies specific for the endostatin oligomers. These antibodies that specifically bind to the endostatin oligomers can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect or quantify the endostatin oligomers in a body fluid or tissue. Results from these tests can be used to diagnose or predict the occurrence or recurrence of a cancer and other angiogenesis mediated diseases. Additionally, passive antibody therapy using antibodies that specifically bind endostatin oligomers can be employed to modulate morphogenic processes such as metastatic cancer as well as angiogenesis-dependent processes such as reproduction, development, wound healing, tissue repair, and angiogenesis-dependent diseases. Also, antisera directed to the Fab I5 regions of endostatin oligomer antibodies can be administered to block the ability of endogenous endostatin oligomer antisera to bind endostatin oligomers.
Antibodies specific for endostatin oligomers may be either polyclonal or monoclonal, and are made according to techniques and protocols well known in the art. The preferred method of making monoclonal antibodies is a modified version of the method of Kearney et al. ( 1979), which is incorporated by reference herein. The monoclonal antibodies are utilized in well known immunoassay formats, such as competitive and non-competitive immunoassays, including ELISA, sandwich immunoassays and radioimmunoassays (RIAs), to determine the presence or absence of the endostatin oligomers of the present invention in body fluids and tissues. Examples of body fluids include, but are not limited to, blood, serum, synovial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterine fluid, saliva, mucus and vitreous humor.
Polyclonal antisera are also raised using established techniques known to those skilled in the art. For example, polyclonal antisera may be raised in mice, rabbits, rats, goats, sheep, guinea pigs, chickens, and other animals, most preferably WO 99/b2944 PCT/US99/12278 mice and rabbits by administering the antigen to the animals.
Isolated, recombinant or synthetic endostatin oligomers conjugated to a carrier molecule such as bovine serum albumin, may be combined with an adjuvant mixture, emulsified and 5 injected subcutaneously at multiple sites on the back, neck, flanks, and sometimes in the footpads of the animals. Booster injections are made at regular intervals, such as every two to four weeks.
Blood samples are obtained by venipuncture, for example using the marginal ear veins after dilation, approximately seven to ten 10 days after each injection. The blood samples are allowed to clot and are centrifuged, and the serum removed, aliquoted, and stored under refrigeration for immediate use or frozen for subsequent analysis.
Techniques for the production of single chain antibodies 15 are known to those skilled in the art and described in U.S. Patent No. 4,946,778 and can be used to produce single chain antibodies to the endostatin oligomers described herein. Bispecific antibodies have two antigen binding domains wherein each domain is directed against a different epitope. Phage display technology may be used to select antibody genes having binding activities for endostatin oligomers from PCR-amplified v genes of lymphocytes from naive libraries or humans screened for having antibodies to the endostatin oligomers.
When labeled with a detectable biomolecule or chemical, the endostatin oligomers and antibodies described above are useful for purposes such as in vivo and in vitro diagnostics and laboratory research using the methods and assays described below. Various types of labels and methods of conjugating the labels to the endostatin oligomers and antibodies are well known to those skilled in the art. Several specific labels are set forth below.
For example, the endostatin oligomers and antibodies are conjugated to a radiolabel such as, but not restricted to, 32P, 3H, 14C, 355, 1251, or 131I. Detection of a label can be by methods such as scintillation counting,, gamma ray spectrometry o r autoradiography.
Bioluminescent labels, such as derivatives of firefly Iuciferin, are also useful. The bioluminescent substance is covalently bound to the endostatin oligomer or antibody by conventional methods, and the labeled endostatin oligomer or antibody is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light.
Fluorogens may also be used as labels. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, alto-phycocyanin, phycocyanin; rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector.
The endostatin oligomers and antibodies can alternatively be labeled with a chromogen to provide an enzyme or affinity label. For example, the endostatin oligomer or antibody can be biotinylated so that it can be utilized in a biotin-avidin reaction; which may also be coupled to a label such as an enzyme or fluorogen. Alternatively, the endostatin oligomer or antibody can be labeled with peroxidase, alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate. Additives such as 5-amino-2,3-dihydro-1,4-phthalazinedione (also known as LuminolTM) (Sigma Chemical Company, St. Louis, MO) and rate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol) (Sigma Chemical Company, St. Louis, MO) can be used to amplify enzymes such as horseradish peroxidase through a luminescent reaction; and luminogenic or fluorogenic dioxetane derivatives of enzyme substrates can also be used. Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer. In addition, endostatin oligomers o r antibodies may be labeled with colloidal gold for use in immunoelectron microscopy in accordance with methods well known to those skilled in the art.
WO 991b2944 PCT/US99/12278 The proteins and antibodies of the present invention are useful for diagnosing and treating metastatic and angiogenesis-related diseases. One example of metastatic disease is metastatic cancer. Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;
rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, ru.beosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. The Endostatin- oligomers of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Hedobacter pylori).
According to the present invention, endostatin oligomers may be used in combination with other compositions and procedures for the treatment of the above described diseases and conditions. For example, a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with endostatin oligomers and subsequently endostatin oligomers may be administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor.
The endostatin oligomers described above can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art.
These formulations can be administered by standard routes. In 1g general, the combinations may be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route. In addition, the endostatin oiigomers may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the endostatin oligomer is slowly released systemically. Osmotic minipumps may also be used to provide controlled delivery of high concentrations of endostatin oligomers through cannuiae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor. The biodegradable polymers and their use are described, for example, in detail in Brem et al. (1991), which is hereby incorporated by reference in its entirety.
The dosage of an endostatin oligomer of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. For treating humans or animals, between approximately 0.5 mg/kilogram to 500 rng/kilogram of the endostatin oligomer and peptide can be administered. A more preferable range is 1 mg/kilogram to 100 mglkilogram with the most preferable range being from 2 mglkilogram to 50 mg/kilogram. Depending upon the half-life of the endostatin oligomer and peptide in the particular animal or human, it can be administered between several times per day to once a week. It is to be understood that the present invention has application f or both human and veterinary use. The methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
The endostatin oligomer formulations include those suitable for oral, rectal, ophthalmic (including intravitreal or WO 99/62944 PCTlUS99/12278 l xntracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural) administration. The endostatin oligomer formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carriers) or excipient(s): In general, the l0 formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary; shaping the product.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats. and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or mufti-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use: Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
Tn addition to methods of treatment, the present invention also relates to methods of using endostatin oligomers, and active i fragments thereof, and antibodies that bind specifically to endostatin oligomers and their peptides, to diagnose endothelial cell-related diseases and disorders. Diagnosis is accomplished by detection of endostatin oligomers, or antibodies thereto, in body 5 fluids or tissues. Detection may be accompanied by comparison of the detected levels of endostatin oligomers, or antibodies thereto, to normal levels of endostatin oligomers, or antibodies thereto.
Kits for measurement of endostatin oligomers are also contemplated as part of the present invention. Antisera that 10 possess the highest titer and specificity and can detect endostatin oligomers in extracts of plasma, urine, tissues, and in cell culture media are further examined to establish easy to use kits for rapid, reliable, sensitive, and specific measurement and localization of endostatin oligomers. These assay kits include but are not limited 15 to the following techniques; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or 20 dipsticks for rapid monitoring of urine or blood, and immunocytochemistry. For each kit, the range, sensitivity, precision, reliability, specificity and reproducibility of the assay is established. Intra-assay and inter-assay variation is established at 20%, SO% and $0% points on the standard curves of displacement or activity.
The assay kit provides instructions, antiserum, endostatin oligomers and possibly radiolabeled endostatin oiigomers or peptides and/or reagents for precipitation ~ of bound antibody complexes. The kit is useful for the measurement of endostatin oligomers in biological fluids and tissue extracts of animals and humans with and without tumors or angiogenesis-dependent diseases.
Also included in the present invention are kits for the localization of endostatin oligomers or peptides in tissues and cells. This endostatin oligomer immunohistochemistry kit WO 99!62944 PCT/US99/122'78 provides instructions, endostatin oligomer antiserum, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum.
Immunohistochemistry techniques are well known to those skilled in the art. This endostatin oligomer and peptide immunohistochemistry kit permits localization of endostatin oligomers in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical l0 purposes. For example, tumors are biopsied or collected and tissue sections cut with a microtome to examine sites of hyaluronic acid binding link module production. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer.
IS The invention further encompasses a method for identifying and quantitating endostatin oligomer-specific receptors. Labeling of the endostatin oligomers with short lived isotopes enables visualization of receptor binding sites in vivo using positron emission tomography or other modern 20 radiographic techniques. These methods are important for the study of angiogenesis and metastasis in cancer and other angiogenesis-related diseases.
This invention is further illustrated by the following examples, which are not to be construed in any way as imposing 25 limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present 30 invention and/or the scope of the appended claims.
Example 1 Production of Endostatin Monomers and Dimers and Oligomers To produce endostatin monomers, the polymerase chain 35 reaction {PCR) was used to adapt the endostatin cDNA for expression in the pdCs-Fc(D4K) vector, which contains the enterokinase recognition site Asp4-Lys at the junction of the fusion protein. The forward primer was 5'-C AAG CTT CAC
AGC CAC CGC GAC TTC C (SEQ ID N0:3), containing a Hind III site followed by a sequence encoding the N-terminus of endostatin (HSHRDFQPVLHL, SEQ ID N0:4}. The reverse primer was 5'-C CTC GAG CTA CTT GGA GGC AGT CAT G, (SEQ ID N0:5} which was designed to put a translation STOP
codon (anti-codon, CTA) immediately after the C-terminus of endostatin, and this was followed by a Xho 1 site (CTCGAG}.
The PCR product was cloned and sequenced, and the Hind III-Xho 1 fragment encoding endostatin was ligated to the pdCsFc(D4K) vector. Stable clones expressing Fc(D4K)-endostatin were obtained by electroporation of NSIO
cells followed by selection in growth medium containing 100 nM
methotrexate. Protein expression levels were assayed by anti-human Fc ELISA as described in Lo, K. M. et al. ( 1998) Protein Engineering 11:495-500 and confirmed by SDS-PAGE.
The best producing clone was sub-cloned by limiting dilutions.
The Fc-human endostatin fusion protein was purified on Protein A sepharose using 100mM citric acid, pH=3.0 as elution buffer. Enterokinase and trypsin digestions were carried out resulting in two forms of cleaved endostatin. For trypsin, the ratio of enzyme to protein (w/w) was 1:200, 18 hours room temperature resulting in an endostatin molecule with four amino acids, HSHR, cleaved from N-terminus. Enterokinase digestion led to a product with one additional amino acid, leucine, at the N-terminus of endostatin. Both cleaved products were purified further using heparin Sepharose and SP sepharose (Pharmacia, Bridgewater, New Jersey).
Disulfide-bonded human endostatin dimer was produced by replacing the restriction fragment encoding the N-terminal sequence of human endostatin in pdCs-Fc(D4K)-hu Endo with a double-stranded oligonucleotide duplex encoding the same sequence but with the Q7 codon changed to C to generate pd WO 99/62944 PCTlUS99/12278 ' 23 Cs-Fc(D4K)-hu Endo (C7): This plasmid was transfected into myeloma cells and the Fc-endostatin dimer was purified from conditioned media utilizing protein A affinity chromatography as described in Lo, K.M. et al.; (1998) Protein Engineering 11:495-500. After the Fc-endostatin dimer was cleaved with enterokinase the endostatin dimer resolved from the Fc portion by gel filtration over S-200.
The endostatin-NCI trimer was produced as follows. The NC I protein was produced using two primers, 5' GAT CGG CCC
1o AGC CGG CCC ATC ATC ACC ATC ACC ATG ACG ATG
ACG ATA AGT CAG GGC AGG TGA GGC TCG CTA CAC
GCA GG 3' (SEQ ID NO:6) and 5'GAT CGG ATC CCT ACT
TGG AGG CAG TCA TGA AGG 3' (SEQ ID N0:7). These primers were used to amplify from the SGQVRLWATRQ amino acid sequence (SEQ ID N0:8} through the C terminus of human collagen I8 using PCR. The PCR products were cloned in the Sfil-BamHl site in pSecTag2a (Invitrogen, San Diego, California}.
Stable 293T cell transfectants were selected in zeocin and conditioned media eluted from Ni-agarose (QIAGEN, Valencia, California) with 250 mM imidazole/PBS followed by dialysis into PBS.
Example 2 Zinc-Dependent Dimers Observed in Crystals of Human Endostatin The X-ray structure was determined from crystals obtained at 4° C by hanging drop vapour diffusion, with equal volumes of 10 mg/ml protein in 20 mM Tris-HCl pH 8.5, 150 mM NaCI and 50 mM Tris-HCI pH 8.5, 6% PEG 8K, 150 mM NaCI and 2 mM
MgC 1 mixed at room temperature in the drops, equilibrated against 0.5 ml of the later buffer. Crystals (C2 a=92.76 t~, b=74.27 ~, c=137.80 ~, 13=102.56 have 4 monomers in the asymmetric unit. Crystals were transferred to 15% PEG 6K, 20%
glycerol, 50 mM Tris-HC 1 pH 8.5 and 150 mM NaCI for about 20 seconds before flash cooling in a stream of cold nitrogen. Data were collected at the CHESS Al station (~,=0.92A) with the 80 mm 2K CCD (bined mode) (0.5 degree oscillations). Data were integrated and scaled with DENZO and SCALEPACK (HKL
Research, Charlottesville, Virginia). Most of the subsequent processing used the CCP4 programs (T. A. Jones ( 1992) "Molecular Replacement" CCP4, 92-105; G. J. Kleywegt and T. A. Jones { 1994) "From First Map to Final Nlodel " CCP4, 59-66).
Following the above methods it was determined that the zinc binding site of human endostatin is tetrahedral with three zinc Iigands from the N-terminal loop, histidines 1, 3, and 11, and a fourth ligand, aspartic acid 76, from the loop between the E and F 13-strands (Fig. 1 and Fig. 2). The zinc binding site most closely resembles that from zinc proteases. Atomic absorption spectroscopy confirmed that zinc is a constituent of the Fe-endostatin chimeras described above of both human and murine endostatin in solution as shown below in Table I.
Table I
Protein N-terminusEndostatin Zinc Zinc per Endostatin sequence ConcentrationConcentrationMonomer (mg/mL) (micromolar) hE-Ek LHSHRD 4 176 0.9 hE-Tryp D 3.8 14 0.1 hFc-hE 0.4 18 0.9 2 79 0.9 The first line of Table I also demonstrates that enterokinase treated Fc-human endostatin; which yields full length endostatin, also showed approximately one atom of zinc per endostatin monomer. Trypsin cleaved Fc-human endostatin, which results in Ioss of the first four residues of endostatin, contains no detectable zinc (second line of Table I). These results are consistent with the human endostatin crystal structure that shows histidines at residues I and 3 to be two of the zinc ligands.
A number of two-fold symmetric dimers are evident in the packing of the four endostatin monomers in the asymmetric unit 5 of the human endostatin crystals. One dimer interface is formed by the contact of two solvent exposed phenylalanines on the a I cc helix from each monomer, a prominent non-polar patch suggested as a potential inter-domain interaction site in murine endostatin (residues F31 and F34 in Fig. 3). Another dimeric contact, that 10 would be zinc dependent, is formed between the projecting N-terminal loops of two monomers; each loop ordered around a zinc ion (Center in Fig. 3a). This contact is formed primarily by three residues that project from the rim of the N-terminal loop:
arginine 4, phenylalanine 6, and glutamine 7 (Fig. 3b). The 15 glutamines at residue 7 of each monomer contact each other forming a hydrogen bond at the center of the interface. Each ring of the phenylalanines at residue 7 contacts a non-polar patch containing Ieucine 78 and valine 72 of the adjacent monomer.
Arginine 4 from each monomer forms hydrogen bonds with two 20 main chain carbonyl oxygens on the loop containing the aspartic acid zinc ligand at residue 76 (carbonyls at residue 75 and 76) (Fig. 3b). This dimeric interaction could only occur if the N-terminal loops were ordered as the result of binding zinc, and in turn, the zinc binding would probably be stabilized by the 25 dimer interface.
Example 3 Zinc Binding of Endostatin is Essential for Anti-Angiogenic Activity As described in Example 2, it was determined that histidines l, 3, 11 and aspartic acid 76 coordinate the zinc ion.
Based upon this discovery, point mutations were introduced into the endostatin protein to change the Zn2+ ligands to alanines. One double (H1/3A) and two single (Hl I A and D76A) mutants were 3S created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California) in an E. coli expression vector.
The mutations were confirmed by sequence analysis.
Wild-type endostatin and the three mutants were purified in parallel and tested in the Lewis lung carcinoma model as previously described in 0'Reilly, M. S. et al., Cell 88:277 (1997) and T. Boehm et al. Nature 390:404 (1997). The different endostatin preparations were compared on a sodium dodecyl sulfate (SDS) gel under reducing and non-reducing conditions and no significant difference in the purity and cross-linking pattern IO was observed. SDS-PAGE of endostatin purified from E. coli showed a protein ladder including monomers, dimers, trimers and higher molecular weight complexes caused by cross-linking of endostatin to other endostatin molecules via disulfide bond formation.
Unlike the wild-type endostatin, none of the three mutants of endostatin regressed Lewis lung carcinoma (data not shown).
Although the . Hl/3A endostatin mutant was weakly active, the weak activity might be due to either a distorted conformation caused by using two histidines from the His.-tag to coordinate the Zn 2+ ion, or by the loss of the ability to bind zinc.
Example 4 Scatter of Pre formed Endothelial Structures by Endostatin Dimers In order to determine the ability of endostatin monomers and dimers to influence morphogenesis, a matrigel tube-formation assay was used. The assay utilizes endothelial cells plated on extracellular matrix proteins that spontaneously aggregate and assemble into densely rnulti-cellular capillary-like tubular structures. The assay was performed as follows.
HUVEC (human umbilical vein endothelial cells), passage <10, isolated from single donor (Clonetics, San Diego, California), were cultured in EBM media (Clonetics, San Diego, California) supplemented with EGM-2-MV growth factors including 5% FCS, hVEGF, hFGF, ( 10 ng/ml), hydrocortisone ( 1 WO 99/62944 PCT/US99/t2278 ~Cg/ml), and GA-1000 (0.1%) at 37° C, 5% CO2, Cells were plated in 1 ml at 40,000 cells/well into 24 well plates containing complete media as above. These wells had been pre-coated at 4° C
with matrigel, a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse -sarcoma (Becton DickinsonJCollaborative, Franklin Lakes, New Jersey) at 300 ,ul/well. Human endostatin derivatives in PBS
( 10-2000 nM) were added either at the time of addition of the cells to the matrigei-coated plates, or after 16 hours of tube formation. Recombinant hHGF (R&D Systems, Minneapolis, Minnesota) was added at 50 ng/ml.
For inhibition of endostatin oligomers by endostatin monomers, pre-formed tubes were pre-incubated with endostatin monomer (2000 nM) for 30 minutes prior to oligomer treatment.
Wells were subsequently incubated at 37° C, 5% CO2, and then photographed under phase contrast microscopy at 40-200x.
Identical results and procedures have been obtained with human microvascular, pulmonary and umbilical artery primary endothelial cultures.
An antibody Fc-endostatin fusion protein containing endostatin at the C-terminus potently inhibited tube formation when included at the time of plating with cells actually becoming more dispersed over time rather than coalescing into tubular structures (data not shown). Identical results were observed with primary cultures of human microvascular, umbilical vein, umbilical artery and pulmonary artery endothelial cells (data not shown). The rapidity of this effect argued against a primary effect on cell growth, and cell counts, BrdU staining and 3H
incorporation did not reveal significant stimulation of proliferation versus untreated cells during this time interval (data not shown).
Intriguingly, after enterokinase cleavage of the Fc-endostatin fusion into Fc and endostatin, neither Fc nor endostatin singly, nor the combination of Fc and endostatin demonstrated inhibitory activity (Figure 4). Recombinant endostatin from baculovirus and yeast un-fused to Fc were also ineffective (data not shown). However, an artificially dimerized endostatin created by utilizing a close contact between adjacent glutamine-7 residues predicted by the dimeric crystal structure of endostatin, was found to exhibit scatter activity. Mutation of endostatin giutamine-7 to cysteine and fusion to antibody Fc allowed formation of a novel intermolecular cysteine bond at cysteine-7, with enterokinase cleavage liberating the free endostatin dimer migrating at 40 kDa and 20 kDa under non-reducing and reducing conditions respectively (data not shown). Both mouse and human endostatin dimers potently reproduced the inhibitory activity of the Fc-endostatin fusion protein on tube formation with ICso of approximately 10 nM (Figure 4) and were inactive against bovine capillary endothelial cell or HUVEC proliferation (data not shown).
During endostatin dimer-induced scattering, rapidly demarginating cells appeared as early as 1-2 hours with prominent lamellipod and pseudopod formation (data not shown).
Confocal microscopy with phalloidin-FITC revealed a dramatic reorganization of the actin cytoskeleton within two hours with prominent stress fiber formation (data not shown), consistent with a signaling pathway linked to rapid actin polymerization. As visualized by electron microscopy, endostatin oligomer-induced scattering was accompanied by destruction of tubular lumens and by cellular elongation (data not shown).
The scatter factor activity of the endostatin oligomers described herein is specific. Although collagen XV (c 15) and collagen XVIII, from which endostatin monomers are derived, exhibit 60% amino acid identity in the endostatin domain, and share the NC 1 trimerization domain, collagen XV oligomers did not act as scatter factors (Figure 4). Fc fusion protein containing the c I5 endostatin-Iike domain was unable to scatter tubules, in contrast to Fc endostatin dimers, and also could not antagonize the effects of Fc endostatin dimers (data not shown). Fc-angiostatin or monomeric angiostatins also did not inhibit tube formation, WO 99162944 PCT/US99/122'18 consistent with the complete lack of structural homology between endostatin and angiostatin (data not shown). Overall, only oligomeric endostatin compositions displayed scatter activity.
Additionally, pretreatment of established tubules with excess of endostatin monomer ( 1000 nM) strongly blocked the ability of endostatin dimers (20-50 nM) to scatter cells (data not shown). This is potentially consistent with endostatin monomers acting in a dominant negative fashion to block endostatin oligomers, for example, by non-productively occupying a receptor that requires oligomer induced cross-linking for activity.
Example 5 Scatter of Pre formed Endothelial Structures by Endostatin-NCI
Trimers Human and marine NC1 proteins were isolated from transfected 293T cell supernatants as described above in Example 1. Trimerization was confirmed by cross-linking the trimerized complexes and resolving the products by non-reduced denaturing polyacrylamide gel electrophoresis (PAGE). Cross-linking was carried out in the presence of 5mM Ethylene Glycol-bis(Succinimidyisuccinate) (EGS), at room temperature for 20 minutes. The EGS was dissolved in DMSO and was diluted ten fold in PBS. The reaction was stopped by adding Tris at a final concentration of 100mM. The resulting gel produced an approximately 120 kDa species upon EGS cross--linking (data not shown).
The marine and human NCl-endostatin trimers identically inhibited endothelial tube formation with an ICSO of approximately 10 nM (Figure 4), again consistent with dependence of activity on endostatin domain oligomerization. The NCI-endostatin trimer was slightly less effective than the endostatin dimers at higher concentrations, possibly reflecting decreased potency from non-covalent versus covalent oligomerization.
Scattering results obtained with the endostatin/NC 1 trimer were similar to the endostatin dimer. The endostatin/NC 1 trimer induced scattering, rapidly demarginating cells appeared as early as 1-2 hours with prominent lamellipod and pseudopod formation 5 (data not shown). Confocal microscopy with phalloidin-FITC
revealed a dramatic reorganization of the actin cytoskeleton within two hours with pronunent stress fiber formation, consistent with a signaling pathway linked to rapid actin polymerization. As visualized by electron microscopy, endostatin 10 oligomer-induced scattering was accompanied by destruction of tubular lumens and by cellular elongation (data not shown).
Pretreatment of established tubules with excess of endostatin monomer (1000 nM} strongly blocked the ability of the NCI-endostatin trimer {20-50 nM) to scatter cells (data not 15 shown). As stated above, this is potentially consistent with endostatin monomers acting in a dominant negative fashion to block endostatin oligomers; for example, by non-productively occupying a receptor that requires oligomer induced cross-linking for activity.
Example 6 Endostati~c Oligomers Comprise a New Ciass of Scatter Factors The oligomeric endostatin-induced cell scattering and cytoskeletal reorganization were reminiscent of morphogenic changes evoked by Hepatocyte Growth Factor/Scatter Factor (HGF/SF), which with the related Macrophage Stimulatory Protein (MSP) comprises a scatter factor family regulating cell motility, dispersion, proliferation and apoptosis. Although Human Umbilical Vein Endothelial Cells (HUVECs) abundantly express the HGF receptor c-met 16, in contrast to endostatin oligomers, HGF was completely unable to scatter HUVEC tubules (data not shown}. Conversely, while HGF potently dis-aggregated and scattered MDCK and HepG2 cell aggregates cultured on plastic, oligomeric endostatin derivatives were inactive (data not shown).
MSP was also unable to elicit HUVEC scatter (data not shown).
WO 99/62944 PCT/US99/t2278 Notably, it is well established that HGF actually promotes tube formation in collagen matrices as opposed to the anti-tubulogenic effects of endostatin described here. Endostatin oligomers therefore define a novel scatter factor class, both structurally and functionally distinct from: the previously unique HGF/MSP
family.
Modifications and variations of the present method will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.
References Brem, H. et al. (1991). Interstitial chemotherapy with drug polymer implants for the treatment of recurrent gliomas. J:
Neurosurg. 74, 441-46.
Chen, C., Parangi, S., Tolentino, M. J., and Folkman; J. (1995).
A strategy to discover circulating angiogenesis inhibitors generated by human tumors: Cancer Res. 55, 4230-4233.
Coleman, J. E. (1992). Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu. Rev.
Biochem. 61, 897-946.
Cunningham, B. C., Bass, S., Fuh, G., and Wells, J. A. ( 1990).
Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science 250, 1709-1712.
Cunningham, B. C., Mulkerrin, M. G., and Wells, J. A. ( 199I ).
Dimerization of human growth hormone by zinc. Science 253, 545-548.
Folkman, J. ( 1989}. What is .the evidence that tumors are angiogenesis dependent?. J. Natl. Cancer Inst. 82, 4-6.
Good, D. J., Polverini, P. J., Rastinejad, F., Le Beau, M. M., Lemons, R. S., Frazier, W.A., and Bouck, N. P. (1990). A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Nat. Acad. Sci. USA. 87, 6624-6628.
Hanahan, D., Folkman, J. (i996}. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364.
Hohenester, E, Sasaki, T., Olsen, B. R:, and Timpl, R. ( 1998}.
Crystal structure of the angiogenesis inhibitor endostatin at 1.5 ~
resolution. The EMB4 J. 17; 1656-1664.
Hori, A., Sasada, R., Matsutani, E., Naito, K., Sakura, Y., Fujita, T., and Kozai, Y. ( 1991 ). Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor. Cancer Res. 51, 6180-6184.
Kandel, J., Bossy-Wetzel, E., Radvany, F., Klagsburn, M., Folkman, J., and Hanahan, D. ( 1991 ). Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. Cell 66, 1095-1104.
Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and Ferrara, N. (1993). Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362, 841-844.
WO 99162944 PCT/US99/1227$
Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., and Ullrich, A. (1994). Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 367, 576-579.
Murga.ki, Y. et al. (1995). Mouse Co118a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones. Proc. Natl. Acad. Sci. USA 92, 8763-8767.
O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R.
A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. ( 1994). Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79, 315-328.
O'Reilly, M. S., Holmgren, L., Chen, C. and Foikman J, (1996).
Angiostatin induces and sustains dormancy of human primary tumors in mice. Nature Med. 2, 689-692.
O'Reilly, M. S. et al. { 1997): Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285.
Oh, S. P. et al. (1994). Isolation and sequencing of cDNAs for proteins with multiple domains of Gly-Xaa-Yaa repeats identify a distinct family of collagenou proteins.
Parangi, S., O'Reilly, M., Christofori, G., Holmgren, L., Grosfeld, J., Folkman, J., and Hanahan, D: ( 1996).
Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc. Natl. Acad. Sci. USA 93, 2002-2007.
Rastinejad, F., Polverini, P. J., and Bouck, N. P. ( 1989).
Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56, 345-355.
a Rehn, M., Pihlajaniemi, T. (1994}. Alphal(XVIII), a collagen chain with frequent interruptions in the collagenous sequence, a distinct tissue distribution, and homology with type XV collagen.
Proc. Natl. Acad. Sci. USA 91, 4234-4238.
Vallee, B. L. and Auld, D. S. ( 1990). Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647-5659.
to Zetter, B.C. (1998). Angiogenesis and Tumor Metastasis. Annu.
Rev. Med. 49, 387-424.
Claims
Claims We Claim:
1. An isolated protein oligomer, comprising more than one endostatin monomer, wherein the oligomer has scatter factor activity.
2. The isolated protein oligomer of Claim 1, wherein the endostatin monomer is a carboxy-terminal region fragment of collagen XVIII having a molecular weight of approximately 20 kDa as determined by reducing gel electrophoresis and 18 kDa as determined by non-reducing gel electrophoresis.
3. The isolated protein oligomer of Claim 1, wherein the oligomer is a dimer of endostatin monomers.
4. The isolated protein oligomer of Claim 3, further comprising a metal component.
5. The isolated protein oligomer of Claim 4, wherein the metal component is zinc.
6. The isolated protein oligomer of Claim 5, wherein the oligomer has anti-tumorigenic activity.
7. The protein oligomer of Claim 1, wherein the protein oligomer comprises more than one NC1 region fragment of collagen XVIII having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC1 fragment contains an endostatin monomer.
8. The isolated protein oligomer of Claim 7, wherein the oligomer is a trimer.
9. The isolated protein oligomer of Claim 8, further comprising a metal component.
10. The isolated protein oligomer of Claim 9, wherein the metal component is zinc.
11. The isolated protein oligomer of Claim 10, wherein the oligomer has anti-tumor activity.
12. The protein oligomer of Claim 1, wherein the endostatin monomers are fusion proteins.
13. The protein oligomer of Claim 12, wherein the endostatin monomers contain endostatin and the Fc portion of an antibody.
14. A method of inhibiting tubulogenesis comprising, administering to an endothelial cell a tubulogenesis inhibiting amount of a protein oligomer comprising more than one endostatin monomer, wherein the oligomer has scatter factor activity.
19. The method of Claim 18, wherein the protein oligomer is a dimer of endostatin monomers.
20. The method of Claim 19, wherein the protein oligomer further comprises a metal component.
21. The method of Claim 20, wherein the metal component is zinc.
22. The method of Claim 18, wherein the protein oligomer comprises more than one NC1 region fragment of collagen XVIII
having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC1 fragment contains an endostatin monomer.
23. The method of Claim 22, wherein the oligomer is a trimer.
24. The method of Claim 23, wherein the protein oligomer further comprises a metal dimerizing component.
25. The method of Claim 24, wherein the metal dimerizing component is zinc.
26. A method of inhibiting tumorigenesis comprising, administering to an endothelial cell a tumorigenesis inhibiting amount of a protein oligomer comprising more than one endostatin monomer, wherein the oligomer has scatter factor activity.
27. The method of Claim 26, wherein the protein oligomer is a dimer of endostatin monomers.
28. The method of Claim 27, wherein the protein oligomer further comprises a metal component.
29. The method of Claim 28, wherein the metal component is zinc.
30. The method of Claim 26, wherein the protein oligomer comprises more than one NC1 region fragment of collagen XVIII
having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC1 fragment contains an endostatin monomer.
31. The method of Claim 30, wherein the oligomer is a trimer.
32. The method of Claim 31, wherein the protein oligomer further comprises a metal dimerizing component.
38~~~~
33. The method of Claim 32, wherein the metal dimerizing component is zinc.
1. An isolated protein oligomer, comprising more than one endostatin monomer, wherein the oligomer has scatter factor activity.
2. The isolated protein oligomer of Claim 1, wherein the endostatin monomer is a carboxy-terminal region fragment of collagen XVIII having a molecular weight of approximately 20 kDa as determined by reducing gel electrophoresis and 18 kDa as determined by non-reducing gel electrophoresis.
3. The isolated protein oligomer of Claim 1, wherein the oligomer is a dimer of endostatin monomers.
4. The isolated protein oligomer of Claim 3, further comprising a metal component.
5. The isolated protein oligomer of Claim 4, wherein the metal component is zinc.
6. The isolated protein oligomer of Claim 5, wherein the oligomer has anti-tumorigenic activity.
7. The protein oligomer of Claim 1, wherein the protein oligomer comprises more than one NC1 region fragment of collagen XVIII having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC1 fragment contains an endostatin monomer.
8. The isolated protein oligomer of Claim 7, wherein the oligomer is a trimer.
9. The isolated protein oligomer of Claim 8, further comprising a metal component.
10. The isolated protein oligomer of Claim 9, wherein the metal component is zinc.
11. The isolated protein oligomer of Claim 10, wherein the oligomer has anti-tumor activity.
12. The protein oligomer of Claim 1, wherein the endostatin monomers are fusion proteins.
13. The protein oligomer of Claim 12, wherein the endostatin monomers contain endostatin and the Fc portion of an antibody.
14. A method of inhibiting tubulogenesis comprising, administering to an endothelial cell a tubulogenesis inhibiting amount of a protein oligomer comprising more than one endostatin monomer, wherein the oligomer has scatter factor activity.
19. The method of Claim 18, wherein the protein oligomer is a dimer of endostatin monomers.
20. The method of Claim 19, wherein the protein oligomer further comprises a metal component.
21. The method of Claim 20, wherein the metal component is zinc.
22. The method of Claim 18, wherein the protein oligomer comprises more than one NC1 region fragment of collagen XVIII
having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC1 fragment contains an endostatin monomer.
23. The method of Claim 22, wherein the oligomer is a trimer.
24. The method of Claim 23, wherein the protein oligomer further comprises a metal dimerizing component.
25. The method of Claim 24, wherein the metal dimerizing component is zinc.
26. A method of inhibiting tumorigenesis comprising, administering to an endothelial cell a tumorigenesis inhibiting amount of a protein oligomer comprising more than one endostatin monomer, wherein the oligomer has scatter factor activity.
27. The method of Claim 26, wherein the protein oligomer is a dimer of endostatin monomers.
28. The method of Claim 27, wherein the protein oligomer further comprises a metal component.
29. The method of Claim 28, wherein the metal component is zinc.
30. The method of Claim 26, wherein the protein oligomer comprises more than one NC1 region fragment of collagen XVIII
having a molecular weight of approximately 38 kDa under reducing gel electrophoresis such that each NC1 fragment contains an endostatin monomer.
31. The method of Claim 30, wherein the oligomer is a trimer.
32. The method of Claim 31, wherein the protein oligomer further comprises a metal dimerizing component.
38~~~~
33. The method of Claim 32, wherein the metal dimerizing component is zinc.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US8789098P | 1998-06-03 | 1998-06-03 | |
US60/087,890 | 1998-06-03 | ||
US9239398P | 1998-07-10 | 1998-07-10 | |
US60/092,393 | 1998-07-10 | ||
US9879098P | 1998-09-01 | 1998-09-01 | |
US60/7098,790 | 1998-09-01 | ||
PCT/US1999/012278 WO1999062944A2 (en) | 1998-06-03 | 1999-06-03 | Protein oligomer compositions comprising endostatin protein and methods of using the same |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2331370A1 true CA2331370A1 (en) | 1999-12-09 |
Family
ID=27375776
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002331370A Abandoned CA2331370A1 (en) | 1998-06-03 | 1999-06-03 | Protein oligomer compositions comprising endostatin protein and methods of using the same |
Country Status (5)
Country | Link |
---|---|
JP (1) | JP2002517186A (en) |
KR (1) | KR20010052566A (en) |
AU (1) | AU4414099A (en) |
CA (1) | CA2331370A1 (en) |
WO (1) | WO1999062944A2 (en) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6440729B1 (en) | 1995-06-30 | 2002-08-27 | University Of Kansas Medical Center | Treating angiogenesis-mediated diseases with the α2 monomer of type IV collagen |
US6358735B1 (en) | 1995-06-30 | 2002-03-19 | University Of Kansas Medical Center | Method for inhibiting angiogenesis and tumors with the isolated NC1 α3 chain monomer of type IV collagen |
AU761027B2 (en) * | 1998-08-25 | 2003-05-29 | Merck Patent Gmbh | Expression and export of angiostatin and endostatin as immunofusis |
US6992174B2 (en) | 2001-03-30 | 2006-01-31 | Emd Lexigen Research Center Corp. | Reducing the immunogenicity of fusion proteins |
US7524811B2 (en) | 2003-08-29 | 2009-04-28 | Children's Medical Center Corporation | Anti-angiogenic peptides from the N-terminus of endostatin |
ATE401400T1 (en) | 2003-08-29 | 2008-08-15 | Childrens Medical Center | ANTIANGIOGENIC PEPTIDES FOR THE TREATMENT OR PREVENTION OF ENDOMETRIOSIS |
FR2898895B1 (en) * | 2006-03-23 | 2012-04-06 | Univ Reims Champagne Ardenne | CYCLOPEPTIDE WITH ANTI-CANCER ACTIVITY DERIVED FROM TYPE IV COLLAGEN |
BRPI0605212B8 (en) * | 2006-12-12 | 2021-05-25 | Univ Rio De Janeiro | production process of dimerized or oligomerized endostatin, dimerized or oligomerized endostatin and pharmaceutical composition |
EP2270055A4 (en) * | 2008-04-04 | 2011-06-29 | Procell Therapeutics Inc | Cell-permeable endostatin recombinant protein, a polynucleotide encoding the same, and an anti-cancer preparation containing the same as an active component |
EP2561888A1 (en) * | 2011-08-23 | 2013-02-27 | Deutsches Krebsforschungszentrum | Protein comprising NC-1 for treating angiogenesis-related diseases |
EP3940065A1 (en) * | 2013-09-30 | 2022-01-19 | Chugai Seiyaku Kabushiki Kaisha | Method for producing antigen-binding molecule using modified helper phage |
CN115925878A (en) * | 2015-06-05 | 2023-04-07 | 艾比欧公司 | Endostatin fragments and variants for the treatment of fibrosis |
ES2968766T3 (en) | 2018-04-17 | 2024-05-13 | Heidelberg Biotech Gmbh | Means and methods for the treatment of diseases related to angiogenesis, fibrosis and cancer with protein oligomers comprising NC-1-FC |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0857210B1 (en) * | 1995-10-23 | 2003-09-03 | The Children's Medical Center Corporation | Therapeutic antiangiogenic compositions and methods |
-
1999
- 1999-06-03 JP JP2000552154A patent/JP2002517186A/en active Pending
- 1999-06-03 KR KR1020007013734A patent/KR20010052566A/en not_active Application Discontinuation
- 1999-06-03 CA CA002331370A patent/CA2331370A1/en not_active Abandoned
- 1999-06-03 AU AU44140/99A patent/AU4414099A/en not_active Abandoned
- 1999-06-03 WO PCT/US1999/012278 patent/WO1999062944A2/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
WO1999062944A2 (en) | 1999-12-09 |
JP2002517186A (en) | 2002-06-18 |
WO1999062944A3 (en) | 2000-04-06 |
AU4414099A (en) | 1999-12-20 |
KR20010052566A (en) | 2001-06-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU717277B2 (en) | Therapeutic antiangiogenic compositions and methods | |
US7867975B2 (en) | Therapeutic antiangiogenic endostatin compositions | |
US5854205A (en) | Therapeutic antiangiogenic compositions and methods | |
US6174861B1 (en) | Methods of inhibiting angiogenesis via increasing in vivo concentrations of endostatin protein | |
CA2331370A1 (en) | Protein oligomer compositions comprising endostatin protein and methods of using the same | |
JP2003517265A (en) | Expression vectors and cell lines expressing vascular endothelial growth factor D and methods of treating melanoma | |
CA2324624A1 (en) | Metastatin and hyaluronate binding proteins and methods of use | |
AU2004202593B2 (en) | Therapeutic antiangiogenic endostatin compositions | |
CA2235393C (en) | Therapeutic antiangiogenic endostatin compositions and methods of use | |
US20040091465A1 (en) | Therapeutic antiangiogenic compositions and methods | |
MXPA98003147A (en) | Compositions of antiangiogenic therapeutic endostatine and methods of |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Dead |