AU2006201128B2 - Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D - Google Patents
Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D Download PDFInfo
- Publication number
- AU2006201128B2 AU2006201128B2 AU2006201128A AU2006201128A AU2006201128B2 AU 2006201128 B2 AU2006201128 B2 AU 2006201128B2 AU 2006201128 A AU2006201128 A AU 2006201128A AU 2006201128 A AU2006201128 A AU 2006201128A AU 2006201128 B2 AU2006201128 B2 AU 2006201128B2
- Authority
- AU
- Australia
- Prior art keywords
- vegf
- tumor
- cells
- sample
- vegfr
- 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.)
- Ceased
Links
- 108010073919 Vascular Endothelial Growth Factor D Proteins 0.000 title claims description 173
- 206010028980 Neoplasm Diseases 0.000 title claims description 130
- 238000000034 method Methods 0.000 title claims description 39
- 102000009519 Vascular Endothelial Growth Factor D Human genes 0.000 title claims 9
- 238000012216 screening Methods 0.000 title description 9
- 210000001519 tissue Anatomy 0.000 claims description 38
- 210000002889 endothelial cell Anatomy 0.000 claims description 37
- 230000012010 growth Effects 0.000 claims description 23
- 201000001441 melanoma Diseases 0.000 claims description 22
- 201000010099 disease Diseases 0.000 claims description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 13
- 210000001165 lymph node Anatomy 0.000 claims description 13
- 206010061289 metastatic neoplasm Diseases 0.000 claims description 12
- 230000001613 neoplastic effect Effects 0.000 claims description 12
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 claims description 11
- 230000001394 metastastic effect Effects 0.000 claims description 11
- 230000004614 tumor growth Effects 0.000 claims description 11
- 239000008280 blood Substances 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 8
- 210000000481 breast Anatomy 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 206010014733 Endometrial cancer Diseases 0.000 claims description 3
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 3
- 208000002151 Pleural effusion Diseases 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 210000002381 plasma Anatomy 0.000 claims description 3
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 201000009030 Carcinoma Diseases 0.000 claims 1
- 102100038234 Vascular endothelial growth factor D Human genes 0.000 description 160
- 210000004027 cell Anatomy 0.000 description 73
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 59
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 57
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 56
- 108010053100 Vascular Endothelial Growth Factor Receptor-3 Proteins 0.000 description 37
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 33
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 description 33
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 30
- BJHCYTJNPVGSBZ-YXSASFKJSA-N 1-[4-[6-amino-5-[(Z)-methoxyiminomethyl]pyrimidin-4-yl]oxy-2-chlorophenyl]-3-ethylurea Chemical compound CCNC(=O)Nc1ccc(Oc2ncnc(N)c2\C=N/OC)cc1Cl BJHCYTJNPVGSBZ-YXSASFKJSA-N 0.000 description 29
- 102000005962 receptors Human genes 0.000 description 29
- 108020003175 receptors Proteins 0.000 description 29
- 210000004881 tumor cell Anatomy 0.000 description 25
- 210000004204 blood vessel Anatomy 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 20
- 230000033115 angiogenesis Effects 0.000 description 20
- 230000014509 gene expression Effects 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 20
- 238000010186 staining Methods 0.000 description 19
- 239000000523 sample Substances 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 201000011510 cancer Diseases 0.000 description 14
- 210000003038 endothelium Anatomy 0.000 description 14
- 230000004807 localization Effects 0.000 description 13
- 210000001365 lymphatic vessel Anatomy 0.000 description 13
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 13
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 12
- 102100038232 Vascular endothelial growth factor C Human genes 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 238000003364 immunohistochemistry Methods 0.000 description 12
- 230000035168 lymphangiogenesis Effects 0.000 description 12
- 230000005747 tumor angiogenesis Effects 0.000 description 12
- 101000742599 Homo sapiens Vascular endothelial growth factor D Proteins 0.000 description 11
- 210000004072 lung Anatomy 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 10
- 239000003102 growth factor Substances 0.000 description 10
- 238000011160 research Methods 0.000 description 10
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 9
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 9
- 230000002491 angiogenic effect Effects 0.000 description 9
- 210000004899 c-terminal region Anatomy 0.000 description 9
- 230000002792 vascular Effects 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 239000002299 complementary DNA Substances 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 210000003491 skin Anatomy 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 210000001072 colon Anatomy 0.000 description 7
- 238000002955 isolation Methods 0.000 description 7
- 230000001926 lymphatic effect Effects 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- 102000007469 Actins Human genes 0.000 description 6
- 108010085238 Actins Proteins 0.000 description 6
- 108020005544 Antisense RNA Proteins 0.000 description 6
- 102100039064 Interleukin-3 Human genes 0.000 description 6
- 108010002386 Interleukin-3 Proteins 0.000 description 6
- 206010029113 Neovascularisation Diseases 0.000 description 6
- 101150044441 PECAM1 gene Proteins 0.000 description 6
- 108010073925 Vascular Endothelial Growth Factor B Proteins 0.000 description 6
- 102100038217 Vascular endothelial growth factor B Human genes 0.000 description 6
- 239000002870 angiogenesis inducing agent Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000003184 complementary RNA Substances 0.000 description 6
- 210000002257 embryonic structure Anatomy 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 229940076264 interleukin-3 Drugs 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 210000004324 lymphatic system Anatomy 0.000 description 6
- 210000000651 myofibroblast Anatomy 0.000 description 6
- 230000002285 radioactive effect Effects 0.000 description 6
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 206010027476 Metastases Diseases 0.000 description 5
- 102100035194 Placenta growth factor Human genes 0.000 description 5
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 5
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 230000013020 embryo development Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000002991 immunohistochemical analysis Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 230000036210 malignancy Effects 0.000 description 5
- 210000002464 muscle smooth vascular Anatomy 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 230000004862 vasculogenesis Effects 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 4
- 108010082093 Placenta Growth Factor Proteins 0.000 description 4
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 4
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 4
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 4
- 102000016663 Vascular Endothelial Growth Factor Receptor-3 Human genes 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 210000002565 arteriole Anatomy 0.000 description 4
- 210000000621 bronchi Anatomy 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000003511 endothelial effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 230000000492 lymphangiogenic effect Effects 0.000 description 4
- 210000005073 lymphatic endothelial cell Anatomy 0.000 description 4
- 210000001077 lymphatic endothelium Anatomy 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- 230000003076 paracrine Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 210000005166 vasculature Anatomy 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 208000008034 Contagious Ecthyma Diseases 0.000 description 3
- 101100481408 Danio rerio tie2 gene Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 101100481410 Mus musculus Tek gene Proteins 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 102000001708 Protein Isoforms Human genes 0.000 description 3
- 108010029485 Protein Isoforms Proteins 0.000 description 3
- 238000011579 SCID mouse model Methods 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 208000014581 breast ductal adenocarcinoma Diseases 0.000 description 3
- 201000010983 breast ductal carcinoma Diseases 0.000 description 3
- 210000001710 bronchial artery Anatomy 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000002872 contrast media Substances 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 210000004207 dermis Anatomy 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000007901 in situ hybridization Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 206010073095 invasive ductal breast carcinoma Diseases 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 201000005296 lung carcinoma Diseases 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 210000004876 tela submucosa Anatomy 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 230000003966 vascular damage Effects 0.000 description 3
- 210000000264 venule Anatomy 0.000 description 3
- NMWKYTGJWUAZPZ-WWHBDHEGSA-N (4S)-4-[[(4R,7S,10S,16S,19S,25S,28S,31R)-31-[[(2S)-2-[[(1R,6R,9S,12S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,53S,56S,59S,62S,65S,68S,71S,76S,79S,85S)-47-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-18-(4-aminobutyl)-27,68-bis(3-amino-3-oxopropyl)-36,71,76-tribenzyl-39-(3-carbamimidamidopropyl)-24-(2-carboxyethyl)-21,56-bis(carboxymethyl)-65,85-bis[(1R)-1-hydroxyethyl]-59-(hydroxymethyl)-62,79-bis(1H-imidazol-4-ylmethyl)-9-methyl-33-(2-methylpropyl)-8,11,17,20,23,26,29,32,35,38,41,48,54,57,60,63,66,69,72,74,77,80,83,86-tetracosaoxo-30-propan-2-yl-3,4,44,45-tetrathia-7,10,16,19,22,25,28,31,34,37,40,49,55,58,61,64,67,70,73,75,78,81,84,87-tetracosazatetracyclo[40.31.14.012,16.049,53]heptaoctacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-7-(3-carbamimidamidopropyl)-25-(hydroxymethyl)-19-[(4-hydroxyphenyl)methyl]-28-(1H-imidazol-4-ylmethyl)-10-methyl-6,9,12,15,18,21,24,27,30-nonaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29-nonazacyclodotriacontane-4-carbonyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@H](Cc4ccccc4)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](Cc4ccccc4)NC3=O)[C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc3ccccc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N3CCC[C@H]3C(=O)N[C@@H](C)C(=O)N2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(=O)N[C@@H](Cc2c[nH]cn2)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(=O)N[C@@H](C)C(O)=O NMWKYTGJWUAZPZ-WWHBDHEGSA-N 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 101150009958 FLT4 gene Proteins 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 102100026849 Lymphatic vessel endothelial hyaluronic acid receptor 1 Human genes 0.000 description 2
- 101710178181 Lymphatic vessel endothelial hyaluronic acid receptor 1 Proteins 0.000 description 2
- 102000004207 Neuropilin-1 Human genes 0.000 description 2
- 108090000772 Neuropilin-1 Proteins 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 241000700635 Orf virus Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 102000006747 Transforming Growth Factor alpha Human genes 0.000 description 2
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 2
- 108091008605 VEGF receptors Proteins 0.000 description 2
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- WLDHEUZGFKACJH-UHFFFAOYSA-K amaranth Chemical compound [Na+].[Na+].[Na+].C12=CC=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(O)=C1N=NC1=CC=C(S([O-])(=O)=O)C2=CC=CC=C12 WLDHEUZGFKACJH-UHFFFAOYSA-K 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 210000003711 chorioallantoic membrane Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 210000004087 cornea Anatomy 0.000 description 2
- 231100001129 embryonic lethality Toxicity 0.000 description 2
- 201000003908 endometrial adenocarcinoma Diseases 0.000 description 2
- 208000029382 endometrium adenocarcinoma Diseases 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000002752 melanocyte Anatomy 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 230000002297 mitogenic effect Effects 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- DLZKEQQWXODGGZ-KCJUWKMLSA-N 2-[[(2r)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KCJUWKMLSA-N 0.000 description 1
- PCDWFBFHIIKIPM-UHFFFAOYSA-N 3-ethyl-2h-1,3-benzothiazole-2-sulfonic acid Chemical compound C1=CC=C2N(CC)C(S(O)(=O)=O)SC2=C1 PCDWFBFHIIKIPM-UHFFFAOYSA-N 0.000 description 1
- QWVRTSZDKPRPDF-UHFFFAOYSA-N 5-(piperidin-1-ylmethyl)-3-pyridin-3-yl-5,6-dihydro-2h-1,2,4-oxadiazine Chemical compound C1CCCCN1CC(N=1)CONC=1C1=CC=CN=C1 QWVRTSZDKPRPDF-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 1
- 208000009458 Carcinoma in Situ Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 102000003712 Complement factor B Human genes 0.000 description 1
- 108090000056 Complement factor B Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 108091060211 Expressed sequence tag Proteins 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000577540 Homo sapiens Neuropilin-1 Proteins 0.000 description 1
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 1
- 101000742596 Homo sapiens Vascular endothelial growth factor C Proteins 0.000 description 1
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 description 1
- 101000851030 Homo sapiens Vascular endothelial growth factor receptor 3 Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101000742598 Mus musculus Vascular endothelial growth factor D Proteins 0.000 description 1
- 101800000021 N-terminal protease Proteins 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010031071 Orf Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000700639 Parapoxvirus Species 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 102000010752 Plasminogen Inactivators Human genes 0.000 description 1
- 108010077971 Plasminogen Inactivators Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- HYLXOQURIOCKIH-VQVTYTSYSA-N Thr-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N HYLXOQURIOCKIH-VQVTYTSYSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091061763 Triple-stranded DNA Proteins 0.000 description 1
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 101001099854 Xenopus laevis Cellular retinoic acid-binding protein 2 Proteins 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000002399 angioplasty Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 108010028263 bacteriophage T3 RNA polymerase Proteins 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000003123 bronchiole Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000003822 cell turnover Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000031902 chemoattractant activity Effects 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 201000005332 contagious pustular dermatitis Diseases 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000004069 differentiation Effects 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
- 239000000539 dimer Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 108091007231 endothelial receptors Proteins 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 210000004996 female reproductive system Anatomy 0.000 description 1
- 230000003328 fibroblastic effect Effects 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
- 230000004927 fusion Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 201000011066 hemangioma Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000050920 human NRP1 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 201000004933 in situ carcinoma Diseases 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000982 limb bud Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 210000005210 lymphoid organ Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000001466 metabolic labeling Methods 0.000 description 1
- 238000007431 microscopic evaluation Methods 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000011206 morphological examination Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000000754 myometrium Anatomy 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 231100001221 nontumorigenic Toxicity 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 239000002797 plasminogen activator inhibitor Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 201000005825 prostate adenocarcinoma Diseases 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000006884 regulation of angiogenesis Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 208000037803 restenosis Diseases 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000011477 surgical intervention Methods 0.000 description 1
- 210000000106 sweat gland Anatomy 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 210000002978 thoracic duct Anatomy 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000006459 vascular development Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 230000004865 vascular response Effects 0.000 description 1
- 230000007998 vessel formation Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: LUDWIG INSTITUTE FOR CANCER RESEARCH Invention Title: METHODS FOR TREATING, SCREENING FOR, AND DETECTING CANCERS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR D The following statement is a full description of this invention, including the best method of performing it known to me/us: METHODS FOR TREATING, SCREENING FOR, AND DETECTING CANCERS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR D. BACKGROUND OF THE INVENTION 5 All references, including any patents or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the 10 applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents 15 form part of the common general knowledge in the art, in Australia or in any other country. The invention generally relates to a method for treating and alleviating melanomas and various cancers, methods for screening for neoplastic diseases, and a 20 method for promoting and maintaining vascularization of normal tissue. The two major components of the mammalian vascular system are the endothelial and smooth muscle cells. The endothelial cells form the lining of the inner surface of 25 all blood vessels and lymphatic vessels in the mammal. The formation of new blood vessels can occur by two different processes, vasculogenesis or angiogenesis (for review see Risau, W., Nature 386: 671-674, 1997). Vasculogenesis is characterized by the in situ 30 differentiation of endothelial cell precursors to mature endothelial cells and association of these cells to form vessels, such as occurs in the formation of the primary vascular plexus in the early embryo. In contrast, angiogenesis, the formation of blood vessels by growth and 35 branching of pre-existing vessels, is important in later embryogenesis and is responsible for the blood vessel growth which occurs in the adult. Angiogenesis is a physiologically complex process involving proliferation of endothelial cells, degradation of extracellular matrix, branching of vessels and subsequent cell adhesion events. In the adult, angiogenesis is tightly controlled and 5 limited under normal circumstances to the female reproductive system. However angiogenesis can be switched on in response to tissue damage. Importantly solid tumors are able to induce angiogenesis in surrounding tissue, thus sustaining tumor growth and facilitating the formation of metastases (Folkman, J., Nature Med. 1: 27-31, 1995). The molecular mechanisms underlying the complex angiogenic processes are far from being understood. Angiogenesis is also involved in a number of pathologic conditions, where it plays a role or is involved directly in different sequelae of the disease. Some examples include neovascularization associated with various liver diseases, neovascular sequelae of diabetes, neovascular sequelae to hypertension, neovascularization in post-trauma, neovascularization due to head trauma, neovascularization in chronic liver infection (e.g. chronic hepatitis), neovascularization due to heat or cold trauma, dysfunction related to excess of hormone, creation of hemangiomas and restenosis following angioplasty. Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGFa), and hepatocyte growth factor (HGF). See for example Folkman et al., J. Biol. Chem., 267: 10931-10934, 1992 for a review. It has been suggested that a particular family of endothelial cell-specific growth factors, the vascular endothelial growth factors (VEGFs), and their corresponding receptors is primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF/VEGF family, and appear to act primarily via endothelial receptor tyrosine kinases (RTKs) . The PDGF/VEGF family of growth factors -2belongs to the cystine-knot superfamily of growth factors, which also includes the neurotrophins and transforming growth factor-B. Eight different proteins have been identified in the PDGF/VEGF family, namely two PDGFs (A and B), VEGF and five members that are closely related to VEGF. The five members clcselr related to VEGF are: VEGF-B, described in International Patent Application PCT/US96/02957 (WO 96/26736) and in U.S. Patents 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki; VEGF-C or VEGF2, described in Joukov et al., EMBO J., 15: 290-298, 1996, Lee et al., Proc. Natl. Acad. Sci. USA, 93: 1988-1992, 1996, and U.S. Patents 5,932,540 and 5,935,540 by Human Genome Sciences, Inc; VEGF-D, described in International Patent Application No. PCT/US97/14696 (WO 98/07832), and Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548-553, 1998; the placenta growth factor (PlGF), described in Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267-9271, 1991; and VEGF3, described in International Patent Application No. PCT/US95/07283 (WO 96/39421) by Human Genome Sciences, Inc. Each VEGF family member has between 30% and 45% amino acid sequence identity with VEGF. The VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cystine-knot motif. Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties. Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that has been isolated from several sources. Alterative mRNA splicing of a single VEGF gene gives rise to five isoforms of VEGF. VEGF shows highly specific mitogenic activity for endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life (Carmeliet - 3 et al., Nature, 380: 435-439, 1996; Ferrara et al., Nature, 380: 439-442, 1996; reviewed in Ferrara and Davis-Smyth, Endocrine Rev., 18: 4-25, 1997). The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele results in embryonic lethality due to failed development of the vasculature (Carmeliet et al., Nature, 380: 435-439, 1996; Ferrara et al., Nature, 380: 439-442, 1996). The isolation and properties of VEGF have been reviewed; see Ferrara et al., J. Cellular Biochem., 47: 211-218, 1991 and Connolly, J. Cellular Biochem., 47: 219-223, 1991. In addition VEGF has strong chemoattractant activity towards monocytes, can induce the plasminogen activator and the plasminogen activator inhibitor in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeability factor (VPF). VEGF is also chemotactic for certain hematopoetic cells. Recent literature indicates that VEGF blocks maturation of dendritic cells and thereby reduces the effectiveness of the immune response to tumors (many tumors secrete VEGF) (Gabrilovich et al., Blood 92: 4150-4166, 1998; Gabrilovich et al., Clinical Cancer Research 5: 2963-2970, 1999). VEGF-B has similar angiogenic and other properties to those of VEGF, but is distributed and expressed in tissues differently from VEGF. In particular, VEGF-B is very strongly expressed in heart, and only weakly in lung, whereas the reverse is the case for VEGF. This suggests that VEGF and VEGF-B, despite the fact that they are co-expressed in many tissues, may have functional differences. VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins which might interact with cellular retinoic acid-binding protein type I (CRABP-I). Its isolation and characteristics are described in -4detail in PCT/US96/02957 (WO 96/26736), in U.S. Patents 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki and in Olofsson et al., Proc. Natl. Acad. Sci. USA, 93: 2576-2581, 1996. VEGF-C was isolated from conditioned media of the PC-3 prostate adenocarcinoma cell line (CRL1435) by screening for ability of the medium to produce tyrosine phosphorylation of the endothelial cell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cells transfected to express VEGFR-3. VEGF-C was purified using affinity chromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNA library. Its isolation and characteristics are described in detail in Joukov et al., EMBO J., 15: 290-298, 1996. VEGF-D was isolated from a human breast cDNA library, commercially available from Clontech, by screening with an expressed sequence tag obtained from a human cDNA library designated "Soares Breast 3NbHBst" as a hybridization probe (Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548-553, 1998). Its isolation and characteristics are described in detail in International Patent Application No. PCT/US97/14696 (W098/07832) . In PCT/US97/14696, the isolation of a biologically active fragment of VEGF-D, designated VEGF-DANAC, is also described. This fragment consists of VEGF-D amino acid residues 93 to 201 linked to the affinity tag peptide FLAG*. The entire disclosure of the International Patent Application PCT/US97/14696 (WO 98/07832) is incorporated herein by reference. The VEGF-D gene is broadly expressed in the adult human, but is certainly not ubiquitously expressed. VEGF-D is strongly expressed in heart, lung and skeletal muscle. Intermediate levels of VEGF-D are expressed in spleen, ovary, small intestine and colon, and a lower expression occurs in kidney, pancreas, thymus, - 5prostate and testis. No VEGF-D mRNA was detected in RNA from brain, placenta, liver or peripheral blood leukocytes. PlGF was isolated from a term placenta cDNA library. Its isolation and characteristics are described in detail in Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267-9271, 1991. Presently its biological function is not well understood. VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3 is stated to have about 36% identity and 66% similarity to VEGF. The method of isolation of the gene encoding VEGF3 is unclear and no characterization of the biological activity is disclosed. Similarity between two proteins is determined by comparing the amino acid sequence and conserved amino acid substitutions of one of the proteins to the sequence of the second protein, whereas identity is determined without including the conserved amino acid substitutions. A major function of the lymphatic system is to provide fluid return from tissues and to transport many extravascular substances back to the blood. In addition, during the process of maturation, lymphocytes leave the blood, migrate through lymphoid organs and other tissues, and enter the lymphatic vessels, and return to the blood through the thoracic duct. Specialized venules, high endothelial venules (HEVs), bind lymphocytes again and cause their extravasation into tissues. The lymphatic vessels, and especially the lymph nodes, thus play an important role in immunology and in the development of metastasis of different tumors. Unlike blood vessels, the embryonic origin of the lymphatic system is not as clear and at least three different theories exist as to its origin. Lymphatic vessels are difficult to identify due to the absence of known specific markers available for them. -6- Lymphatic vessels are most commonly studied with the aid of lymphography. In lymphography, X-ray contrast medium is injected directly into a lymphatic vessel. The contrast medium gets distributed along the efferent drainage vessels of the lymphatic system and is collected in the lymph nodes. The contrast medium can stay for up to half a year in the lymph nodes, during which time X-ray analyses allow the follow-up of lymph node size and architecture. This diagnostic is especially important in cancer patients with metastases in the lymph nodes and in lymphatic malignancies, such as lymphoma. However, improved materials and methods for imaging lymphatic tissues are needed in the art. As noted above, the PDGF/VEGF family members act primarily by binding to receptor tyrosine kinases. In general, receptor tyrosine kinases are glycoproteins, which consist of an extracellular domain capable of binding a specific growth factor(s), a transmembrane domain, which is usually an alpha helical portion of the protein, a juxtamembrane domain, which is where the receptor may be regulated by, e.g., protein phosphorylation, a tyrosine kinase domain, which is the enzymatic component of the receptor and a carboxy-terminal tail, which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase. Five endothelial cell-specific receptor tyrosine kinases have been identified, namely VEGFR-l (Flt-1), VEGFR-2 (KDR/Flk-1) , VEGFR-3 (Flt4), Tie and Tek/Tie-2. These receptors differ in their specificity and affinity. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction. The only receptor tyrosine kinases known to bind VEGFs are VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-l and VEGFR-2 bind VEGF with high affinity, and VEGFR-l also binds VEGF-B and PlGF. VEGF-C has been shown to be the ligand for VEGFR-3, and it also -7activates VEGFR-2 (Joukov et al., The EMBO Journal, 15: 290-298, 1996). VEGF-D binds to both VEGFR-2 and VEGFR-3 (Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548-553, 1998). A ligand for Tek/Tie-2 has been described in International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie has not yet been identified. Recently, a novel 130-135 kDa VEGF isoform specific receptor has been purified and cloned (Soker et al., Cell, 92: 735-745, 1998). The VEGF receptor was found to specifically bind the
VEGF
165 isoform via the exon 7 encoded sequence, which shows weak affinity for heparin (Soker et al., Cell, 92: 735-745, 1998). Surprisingly, the receptor was shown to be identical to human neuropilin-1 (NP-1), a receptor involved in early stage neuromorphogenesis. PlGF-2 also appears to interact with NP-1 (Migdal et al., J. Biol. Chem., 273: 22272-22278, 1998). VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells. Generally, both VEGFR-l and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., Oncogene, 8: 11-18, 1992; Kaipainen et al., J. Exp. Med., 178: 2077-2088, 1993; Dumont et al., Dev. Dyn., 203: 80-92, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996) and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl. Acad. Sci. USA, 9: 3566-3570, 1995) . VEGFR-3 is also expressed in the blood vasculature surrounding tumors. Although VEGFR-l is mainly expressed in endothelial cells during development, it can also be found in hematopoetic precursor cells during early stages of embryogenesis (Fong et al., Nature, 376: 66-70, 1995). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 87: 3336-3343, 1995) . In embryos, VEGFR-1 is expressed by most, -8if not all, vessels (Breier et al., Dev. Dyn., 204: 228-239, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996). The receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development but as embryogenesis proceeds becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., Cancer Res., 54: 6571 6577, 1994; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570, 1995) . VEGFR-3 is expressed on lymphatic endothelial cells in adult tissues. This receptor is essential for vascular development during embryogenesis. The essential, specific role in vasculogenesis, angiogenesis and/or lymphangiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos. Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation. Analysis of embryos carrying a completely inactivated VEGFR-l gene suggests that this receptor is required for functional organization of the endothelium (Fong et al., Nature, 376: 66-70, 1995) . However, deletion of the intracellular tyrosine kinase domain of VEGFR-l generates viable mice with a normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95: 9349-9354, 1998) . The reasons underlying these differences remain to be explained but suggest that receptor signalling via the tyrosine kinase is not required for the proper function of VEGFR-l. Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoesis and vasculogenesis (Shalaby et al., Nature, 376: 62-66, 1995; Shalaby et al., Cell, 89: 981-990, 1997). Targeted inactivation of both copies of the. VEGFR-3 gene in mice resulted in defective blood vessel formation characterized by abnormally organized large vessels with defective lumens, leading to fluid -9accumulation in the pericardial cavity and cardiovascular failure at post-coital day 9.5 (Dumont et al., Science, 282: 946-949, 1998). On the basis of these findings it has been proposed that VEGFR-3 is required for the maturation of primary vascular networks into larger blood vessels. However, the role of VEGFR-3 in the development of the lymphatic vasculature could not be studied in these mice because the embryos died before the lymphatic system emerged. Nevertheless it is assumed that VEGFR 3 plays a role in development of the lymphatic vasculature and lymphangiogenesis given its specific expression in lymphatic endothelial cells during embryogenesis and adult life. This is supported by the finding that ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice, resulted in lymphatic endothelial cell proliferation and vessel enlargement in the dermis. Furthermore this suggests that VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al., EMBO J., 15: 290-298, 1996). In addition, VEGF-like proteins have been identified which are encoded by four different strains of the orf virus. This is the first virus reported to encode a VEGF-like protein. The first two strains are NZ2 and NZ7, and are described in Lyttle et al., J. Virol., 68: 84-92, 1994. A third is D1701 and is described in Meyer et al., EMBO J., 18: 363-374, 1999. The fourth strain is NZl0 and is described in International Patent Application PCT/US99/25869. It was shown that these viral VEGF like proteins bind to VEGFR-2 on the endothelium of the host (sheep/goat/human) and this binding is important for development of infection (Meyer et al., EMBO J., 18: 363-374, 1999; Ogawa et al. J. Biol. Chem., 273: 31273-31282, 1988; and International Patent Application PCT/US99/25869). These proteins show amino acid sequence similarity to VEGF and to each other. - 10 - The orf virus is a type of species of the parapoxvirus genus which causes a highly contagious pustular dermatitis in sheep and goats and is readily transmittable to humans. The pustular dermatitis induced by orf virus infection is characterized by dilation of blood vessels, swelling of the local area and marked prcliferation of endothelial cells lining the blood vessels. These features are seen in all species infected by orf and can result in the formation of a tumor-like growth or nodule due to viral replication in epidermal cells. Generally orf virus infections resolve in a few weeks but severe infections that fail to resolve without surgical intervention are seen in immune impaired individuals. There is tremendous interest in the development of pharmacological agents which could antagonize the receptor mediated actions of VEGFs (Martiny-Baron and Marme, Curr. Opin. Biotechnol. 6: 675-680, 1995). Monoclonal antibodies to VEGF have been shown to suppress tumor growth in vivo by inhibiting tumor-associated angiogenesis (Kim et al., Nature 362: 841-844, 1993) . Similar inhibitory effects on tumor growth have been observed in model systems resulting from expression of either antisense RNA for VEGF (Saleh et al., Cancer Res. 56: 393-401, 1996) or a dominant-negative VEGFR-2 mutant (Millauer et al., Nature 367: 576-579, 1994). However, tumor inhibition studies with neutralizing antibodies suggested that other angiogenic factors besides VEGF may be involved (Kim, K. et al., Nature 362: 841-844, 1993) . Furthermore, the activity of angiogenic factors other than VEGF in malignant melanoma is suggested by the finding that not all melanomas express VEGF (Issa, R. et al., Lab Invest 79: 417-425, 1999). The biological functions of the different members of the VEGF family are currently being elucidated. Of particular - 11 interest are the properties of VEGF-D and VEGF-C. These proteins bind to both VEGFR-2 and VEGFR-3 - localized on vascular and lymphatic endothelial cells respectively - and are closely related in primary structure (48% amino acid identity) . Both factors are mitogenic for endothelial cells in vitro. Recently, VEGF-C was shown to be angiogenic in the moure cornea model and in the avian chorioallantoic membrane (Cao et al., Proc. Natl. Acad. Sci. USA 95: 14389-14394, 1998) and was able to induce angiogenesis in the setting of tissue ischemia (Witzenbichler et al., Am. J. Pathol. 153: 381-394, 1998). Furthermore, VEGF-C stimulated lymphangiogenesis in the avian chorioallantoic membrane (Oh et al., Dev. Biol. 188: 96-109, 1997) and in a transgenic mouse model (Jeltsch et al., Science 276: 1423-1425, 1997). VEGF-D was shown to be angiogenic in the rabbit cornea (Marconcini et al., Proc. Natl. Acad. Sci. USA 96: 9671-9676, 1999). The lymphangiogenic capacity of VEGF-D has not yet been reported, however, given that VEGF-D, like VEGF-C, binds and activates VEGFR-3, a receptor thought to signal for lymphangiogenesis (Taipale et al., Cur. Topics Micro. Immunol. 237: 85-96, 1999), it is highly likely that VEGF-D is lymphangiogenic. VEGF-D and VEGF-C may be of particular importance for the malignancy of tumors, as metastases can spread via either blood vessels or lymphatic vessels; therefore molecules which stimulate angiogenesis or lymphangiogenesis could contribute toward malignancy. This has already been shown to be the case for VEGF. It is noteworthy that VEGF-D gene expression is induced by c-Fos, a transcription factor known to be important for malignancy (Orlandini et al., Proc. Natl. Acad. Sci. USA 93: 11675-11680, 1996). It is speculated that the mechanism by which c-Fos contributes to malignancy is the upregulation of genes encoding angiogenic factors. Tumor cells deficient in c-fos fail to undergo malignant progression, possibly due to an inability - 12 to induce angiogenesis (Saez, E. et al., Cell 82: 721-732, 1995). This indicates the existence of an angiogenic factor up-regulated by c-fos during tumor progression. As shown in Figure 1, the predominant intracellular 5 form of VEGF-D is a homodimeric propeptide that consists of the VEGF/PDGF Homology Domain (VHD) and the N- and C terminal propeptides and has the sequence of SEQ ID NO:2. After secretion, this polypeptide is proteolytically cleaved (Stacker, S.A. et al., J Biol Chem 274: 32127 10 32136, 1999). Proteolytic processing (at positions marked by black arrowheads) gives rise to partially processed forms and a fully processed, mature form which consists of dimers of the VHD. The VHD, which has the sequence of SEQ ID NO:3 (i.e. residues 93 to 201 of full length VEGF-D), 15 contains the binding sites for both VEGFR-2 and VEGFR-3. The mature form binds both VEGFR-2 and VEGFR-3 with much higher affinity than the unprocessed form (Stacker, S.A. et al., J Biol Chem 274: 32127-32136, 1999) The localization of VEGF-D protein in human cancer 20 has not been studied due to the lack of antibodies specific for the VHD of VEGF-D. Antibodies against the N or C-terminal propeptides are inappropriate as these regions are cleaved from the bioactive VHD and would localize differently than the VHD (Stacker, S.A. et al., J 25 Biol Chem 274: 32127-32136, 1999). SUMMARY OF THE INVENTION The invention generally relates to a method for diagnosing growth characteristics of a tumor or neoplastic 30 disease in an organism. In a first aspect, the invention provides a method of diagnosing growth characteristics of a neoplastic disease in an organism, the method comprising: (a) measuring amount of unprocessed VEGF-D 35 polypeptide in a sample from an organism with a neoplastic disease; and 13 N:\M elb n\CaAs\Patent\46000-46999\P46490-AU.1\Specis\P46490-AU.1 amenents 2009-3-23.doc (b) diagnosing growth characteristics of the neoplastic disease from the amount of the VEGF-D measured in step (a), wherein increased unprocessed VEGF-D in said sample correlates with increased tumor growth or 5 metastatic risk. In a second aspect, the invention provides a method of diagnosing growth characteristics of a tumor in an organism, the method comprising: (a) measuring amount of unprocessed VEGF-D 10 polypeptide in a sample from an organism with a neoplastic disease; and (b) diagnosing growth characteristics of the tumor from the amount of the VEGF-D measured in step (a), wherein increased unprocessed VEGF-D in said sample 15 correlates with increased tumor growth or metastatic risk. The measuring step may comprise exposing said sample to a composition comprising an antibody that specifically binds VEGF-D, wherein said antibody is a monoclonal antibody, and/or wherein said antibody includes a 20 detectable label. In one embodiment of the invention, the unprocessed VEGF-D has an apparent molecular weight of -53K. In a further embodiment of the invention the sample is selected from the group consisting of tissue, blood, 25 serum, plasma, urine, ascities fluid and pleural effusion. Particularly, the sample is a tissue sample, or comprises endothelial cells or a lymph node. In a further embodiment of the invention, the neoplastic disease is selected from the group consisting 30 of malignant melanoma, breast ductal carcinoma, squamous cell carcinoma, prostate cancer and endometrial cancer. In a further embodiment of the invention the organism is a human. It will be clearly understood that for the purposes 35 of this specification the term "sample" includes, but is not limited to, obtaining a tissue sample, blood, serum, 14 N:kMelbourn\Cse\Patent\46000-46999\P46490.AU.1\Specis\P46490.AU.1 amendment 200-3-23.doc plasma, urine, ascities fluid or pleural effusion. Preferably the tissue is human tissue and the compound is preferably a monoclonal antibody. It will be appreciated that use of the second compound helps the practitioner to 5 confirm that the VEGF-D found on the vessels in or near the tumor arises due to receptor-mediated uptake, which supports the hypothesis that VEGF-D, secreted by tumor cells, binds and accumulates in target endothelial cells thereby establishing a paracrine mechanism regulating 10 tumor angiogenesis. Antibodies for use in the invention also may be labeled with a detectable label. The antibody may be covalently or non-covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, ecogenic or 15 radioactive agent for imaging. Radioactive or non radioactive labels may be used. Examples of radioactive labels include a radioactive atom or group, such as 1251 or P. Examples of non-radioactive labels include enzyme labels, such as horseradish peroxidase, or fluorimetric 20 labels, such as fluorescein-5-isothiocyanate (FITC). Labeling may be direct or indirect, covalent or non covalent. In the claims of this application and in the description of the invention, except where the context 25 requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further 30 features in various embodiments of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of VEGF-D 35 processing; 15 N: \Malbourna\Case\Patent\400 -4g6gg\P4g4g A 2 \P4G490.AU. I mendmaeto 2009-3-23.doc Figure 2 shows the specificity of MAb 4A5 for the VEGF/PDGF Homology Domain (VHD) of human VEGF-D as assessed by Western blot analysis; 5 Figure 3 shows autoradiographs taken after two days of exposure to mouse 15.5 days post-coital tissue sections hybridized with VEGF-D antisense and sense RNAs; Figures 4A-4D show the results of analysis of the 10 distribution of VEGF-D mRNA in the post-coital day 15.5 mouse embryo by in situ hybridization; Figures 5A-5H show the results of immunohistochemical analysis from two malignant melanomas exemplifying the 15 different reaction patterns; Figures 6A-6F show the localization of VEGF-D in squamous cell carcinoma of the lung; 16 N\Melbourne\Cases\Patent\46000--46999\P46490.AT.1\SpeciS\P46490.AU.1 almendments 2009-3-23,doc THIS PAGE IS INTENTIONALLY BLANK 17 Nr \Kelboure\cases\Patent\46000-46999\P4649o.AU.L\SpeciB\P46490 .AU.1 amendMentS 2009-3-23.doc THIS PAGE IS INTENTIONALLY BLANK 18 N: \Melborne\Cases\Patent\46000-46999\P46490.AU.\Specis\P4649O.AU.1 amendmentg 2009-3-23.doc THIS PAGE IS INTENTIONALLY BLANK 19 N: \Kelbourne\Cases\Patent\46000-46999\P4649.AU.l\Specis\P46490.AU.1 amendOents 2009-3-23.doc THIS PAGE IS INTENTIONALLY BLANK 20 Nz\Me2bourne\Case\Patent\46000-4699\P46490.AU.l\Specis\P46490,AU.1 amendments 2009-3-23.doC THIS PAGE IS INTENTIONALLY BLANK 21 N\Melbourne\CateS\Patent\46000-46999\P46490.AU.I\Specis\P46490.AU.1 amendments 2009-3-23.doc THIS PAGE IS INTENTIONALLY BLANK 22 N:\Melburne\cases\Patent\ 4 6coo- 4 6 999\P 4 649o.AU I\speCis\P46490.A.3 amendment& 2009- -23.doc THIS PAGE IS INTENTIONALLY BLANK 23 N:\Melbourne\Case\Patent\ 4 6fo-46999\p4gAD.,1\SPci.B\P4490.AU-1 amendments 2009-3-23-doe THIS PAGE IS INTENTIONALLY BLANK 24 N e\elbourne\Casee\Patent\46000-46999\P46490 .AU.ASpecis\P46490.AU.1 amendments 2009-3-23.doc THIS PAGE IS INTENTIONALLY BLANK 25 N:\MeLborne\Cases\Patent\46000-46999\P46 4 9 o.AU.l\SpecieP46490.AU.1 anmn-l-t 2009-3-23.doc THIS PAGE IS INTENTIONALLY BLANK 26 N.\Melbourne\Cases\Patent\46000-46999\p46490.AU.L\Speci;\P46490.mA.L aMwnaenta 2009-3-23.doc THIS PAGE IS INTENTIONALLY BLANK 27 MrMrl~ne\CaseekPatet\t4go..4g,99\N4490.AU.\Specis\N649 0
,AV.
1 amdmets 200 9 -3-23.doc THIS PAGE IS INTENTIONALLY BLANK 28 N.\Melbourne\Cases\Patnt\ 4 6000-4G999\P4 4R0.AU. 1\pmcdw\P4649 0 .AU- am|encklut 2009-3-23.doc THIS PAGE IS INTENTIONALLY B3LANK 29 N. \N~bcurmOxcaBce\Patelat\ 4 6 oo..
4 6 9
;,\
4 6 4 9 0 .Al.1\8pecis\P4$$90.AU.1 amendments 2009-3-23 .doc Figures 7A-7F show the localization of VEGF-D in breast ductal carcinoma in situ; Figure 8 shows the localization of VEGF-D in 5 endometrial adenocarcinoma in situ; Figure 9A-9F show the localization of VEGF-D in normal colon tissue. 10 Figure 10 shows the results of the analysis of tumors in SCID mice resulting from injection of untransfected parental 293 cells (designated "293") and 293 cells transfected with an expression vector encoding VEGF-D- FULL-N-FLAG (designated "VEGF-D-293"). 15 Figure 11 shows a tumor produced by VEGF-DAN cells. Figure 12 shows a normal tumor. 20DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1 Production of monoclonal antibodies that bind to human VEGF-D In order to detect the VEGF/PDGF Homology Domain (VHD) 25rather than the N- and C-terminal propeptides, monoclonal antibodies to the mature form of human VEGF-D (residues 93 to 201 of full-length VEGF-D (SEQ ID NO:2), i.e. with the N- and C terminal regions removed) were raised in mice. A DNA fragment encoding residues 93 to 201 was amplified by polymerase chain 30reaction (PCR) with Pfu DNA polymerase, using as template a plasmid comprising full-length human VEGF-D cDNA (SEQ ID NO:1) . The amplified DNA fragment, the correctness of which was - 30 confirmed by nucleotide sequencing, was then inserted into the expression vector pEFBOSSFLAG (a gift from Dr. Clare McFarlane at the Walter and Eliza Hall Institute for Medical Research (WEHI), Melbourne, Australia) to give rise to a plasmid 5designated pEFBOSVEGF-DANAC. The pEFBOSSFLAG vector contains DNA encoding the signal sequence for protein secretion from the interleukin-3 (IL-3) gene and the FLAG* octapeptide (Sigma-Aldrich) The FLAG octapeptide can be recognized by commercially available antibodies such as the M2 monoclonal 10antibody (Sigma-Aldrich) . The VEGF-D PCR fragment was inserted into the vector such that the IL-3 signal sequence was immediately upstream from the FLAG* octapeptide, which was in turn immediately upstream from the truncated VEGF-D sequence. All three sequences were in the same reading frame, so -that 15translation of mRNA resulting from transfection of pEFBOSVEGF DANAC into mammalian cells would give rise to a protein which would have the IL-3 signal sequence at its N-terminus, followed by the FLAG® octapeptide and the truncated VEGF-D sequence. Cleavage of the signal sequence and subsequent secretion of the 20protein from the cell would give rise to a VEGF-D polypeptide which is tagged with the FLAG® octapeptide adjacent to the N terminus. VEGF-DANAC was purified by anti-FLAG® affinity chromatography from the medium of COS cells which had been transiently transfected with the plasmid pEFBOSVEGF-DANAC. (see 25Example 9 in International Patent Application No. PCT/US97/14696). Purified VEGF-DANAC was used to immunize female Balb/C mice on day 85 (intraperitoneal), 71 (intraperitoneal) and 4 (intravenous) prior to the harvesting of the spleen cells from 30the immunized mice and subsequent fusion of these spleen cells to mouse myeloma P3X63Ag8.653 (NS-1) cells. For the first two immunizations, approximately 10 pg of VEGF-DANAC in a 1:1 mixture - 31 of PBS and TiterMax adjuvant (#R-1 Research adjuvant; CytRx Corp., Norcross, GA) were injected, whereas for the third immunization 35 pg of VEGF-DANAC in PBS was used. Monoclonal antibodies to VEGF-DANAC were selected by screening the hybridomas on purified VEGF-DANAC using an enzyme immunoassay. Briefly, 96-well microtiter plates were coated with VEGF-DANAC, and hybridoma supernatants were added and incubated for 2 hours at 4*C, followed by six washes in PBS with 0.02% Tween 20. Incubation with a horse radish peroxidase conjugated 10anti-mouse Ig (Bio-Rad, Hercules, CA) followed for 1 hour at 4 0 C. After washing, the assay was developed with an 2,2'-azino-di- (3 ethylbenz-thiazoline sulfonic acid) (ABTS). substrate system (Zymed, San Francisco, CA), and the assay was quantified by reading absorbance at 405 nm in a multiwell plate reader (Flow 15Laboratories MCC/340, McLean, VA). Six antibodies were selected for further analysis and were subcloned twice by limiting dilution. These antibodies were designated 2F8, 3C10, 4A5, 4E10, 4H4 and 5F12. The is-otypes of the antibodies were determined using an Isostrip" isotyping kit (Boehringer Mannheim, 20Indianapolis, IN) . Antibodies 2F8, 4A5, 4E10 and 5F12 were of the IgG 1 class whereas 4H4 and 3C10 were of the IgM class. All six antibodies contained the kappa light chain. Hybridoma cell lines were grown in DMEM containing 5% v/v IgG-depleted serum (Gibco BRL, Gaithersburg, MD), 5mM L 25glutamine, 50 pg/ml gentamicin and 10 pg/ml recombinant IL-6. Antibodies 2F8, 4A5, 4E10 and 5F12 were purified by affinity chromatography using protein G-Sepharose according to the technique of Darby &t al., J. Immunol. Methods 159: 125-129, 1993, and the yield assessed by measuring absorption at 280nm. 30 - 32 - Example 2 Specificity of 4A5 The specificity of MAb 4A5 (renamed VD1) for the VHD of human VEGF-D was assessed by Western blot analysis. Derivatives of VEGF-D used were VEGF-DANLC, consisting of amino acid residues 593 to 201 of human VEGF-D tagged at the N-terminus with the FLAG* octapeptide (Example 1) , VEGF-D-FFULL-N-FLAG, consisting of full length VEGF-D tagged at the N-terminus with FLAG* (Stacker, S.A. et al., J Biol Chem 274: 32127-32136, 1999), and VEGF-D-CPRO, consisting of the C-terminal propeptide, from amino acid residues 10206 to 354, which was also tagged with FLAG* at the N-terminus. These proteins were expressed in 293-EBNA-1 cells, purified by affinity chromatography with M2 (anti-FLAG) MAb (IBI/Kodak, New Haven, CT) using the procedure set forth in Achen, M. et al., Proc Natl Acad Sci USA 95: 548-553, 1998. Fifty nanograms of 15purified VEGF-D-FULL-N-FLAG (FN), VEGF-DANAC (AA), and VEGF-D CPRO (CP) were analyzed by SDS-PAGE (reducing) and by Western blot using the VD1 MAb and a biotinylated M2 MAb as control (the antibody used for blotting is indicated at the bottom of the panel of Figure 2) . SDS-Page and Western blot analyses were 20carried out as described in Stacker, S.A. et al., J Biol Chem 274: 32127-32136, 1999. As shown in Figure 2, the predominant species in the sample of VEGF-D-FULL-N-FLAG consist of unprocessed VEGF-D (Mr -53 K), partially processed VEGF-D containing both the N-terminal 25propeptide and the VHD (-31 K) , and the N-terminal propeptide (-10 K) (Stacker, S.A. et al., J Biol Chem 274: 32127-32136, 1999), all of which are detected with the M2 MAb as they are tagged with the FLAG* octapeptide (arrows to the left, numbers represent Mr in K and subscripts indicate the sample in which the 30band is detected) . Likewise, VEGF-DANAC (-21 K) and VEGF-D-CPRO (two bands of -31 and -29 K which arise due to differential glycosylation) are detected with M2 (arrows to the left) as these - 33 polypeptides are also tagged with FLAG*. VD1 detects unprocessed VEGF-D, partially processed VEGF-D and VEGF-DANAC, but not the N-terminal propeptide (-10 K) in the VEGF-D-FULL-N-FLAG preparation, nor the C-terminal propeptide in the VEGF-D-CPRO sample (-31 and -29 K). Results with VEGF-D-FULL-N-FLAG were analyzed with long (L) and short (S) exposures. The positions of molecular weight markers are shown to the right in Figure 2. Thus MAb VD1 binds unprocessed VEGF-D, partially processed forms containing the VHD and fully processed VEGF-D, but not the 1ON- or C-terminal propeptides. Furthermore, MAb VD1 was able to immunoprecipitate native human VEGF-DANAC, but not the VHD of human VEGF-C (VEGF-CNAC) (Joukov, V. et al., EMBO J 16: 3898 3911, 1997) in an enzyme immunoassay indicating that VD1 is specific for VEGF-D. 15 Example 3 In Situ Hybridization Studies of VEGF-D Gene Expression in Mouse Embryos The pattern of VEGF-D gene expression was studied by in situ hybridization using a radiolabeled antisense RNA probe 20corresponding to nucleotides 1 to 340 of the mouse VEGF-D1 cDNA (SEQ ID NO:4). The antisense RNA was synthesized by in vitro transcription with T3 RNA polymerase and [ 35 S]UTPas. Mouse VEGF-D is fully described in International Patent application PCT/US97/14696 (WO 98/07832) . This antisense RNA probe was 25hybridized to paraffin-embedded tissue sections of mouse embryos at post-coital day 15.5. The labeled sections were subjected to autoradiography for 2 days. The resulting autoradiographs for sections hybridized to the antisense RNA and to complementary sense RNA (as negative control) are shown in Figure 3. In Figure 303, "L" denotes lung and "Sk" denotes skin, and the two tissue sections shown are serial sections. Strong signals for VEGF-D - 34 mRNA were detected in the developing lung and associated with the skin. No signals were detected using the control sense RNA. In Figures 4A-4D, sagittal tissue sections were hybridized with the VEGF-D antisense RNA probe and subsequently incubated with photographic emulsion, developed and stained. The magnification for Figures 4A and 4D is x40, for Figure 4B, it is x200 and for Figure 4C, it is x500. In Figure 4A, the dark field micrograph shows a strong signal for VEGF-D mRNA in lung (Lu). Liver (Li) and ribs (R) are 10also shown. Figure 4B shows a higher magnification of the lung. This light field micrograph shows a bronchus (Br) and bronchial artery (BA). The black outline of a rectangle denotes the region of the section shown in Figure 4C but at a higher magnification. Figure 4C shows the epithelial cells of the bronchus (Ep), the 15developing smooth muscle cells (SM) surrounding the epithelial cell layer and the mesenchymal cells (Mes) . The abundance of silver grains associated with mesenchymal cells is apparent. -Thus, microscopic analysis reveals that VEGF-D mRNA is abundant in the mesenchymal cells of the developing lung (Figures 4A-4C). 20In contrast, the epithelial cells of the bronchi and bronchioles are negative, as were the developing smooth muscle cells surrounding the bronchi. The endothelial cells of bronchial arteries are also negative. In Figure 4D, a dark field micrograph shows a limb bud. A 25strong signal was located immediately under the skin in a region of tissue rich in fibroblasts and developing melanocytes. These results indicatethat VEGF-D may attract the growth of blood and lymphatic vessels into the developing lung and into the region immediately underneath the skin. Due to the 30expression of the VEGF-D gene adjacent to embryonic skin, it is considered that VEGF-D could play a role in inducing the angiogenesis that is associated with malignant melanoma. - 35 - Malignant melanoma is a very highly vascularized tumor. This suggests that local inhibition of VEGF-D expression, for example using VEGF-D or VEGF receptor-2 or VEGF receptor-3 antibodies, is useful in the treatment of malignant melanoma. Other suitable Inhibitors of VEGF-D activity, such as anti-sense nucleic acids or triple-stranded DNA, may also be used. Example 4 Use of monoclonal antibodies to human VEGF-D for immunohistochemical analysis of human tumors 10 In order to assess the role of VEGF-D in tumor angiogenesis, VEGF-D MAbs, 4A5, 5F12 and 2F8 (renamed VD1, VD2 and VD3, respectively) were used for immunohistochemical analysis of fifteen randomly chosen invasive malignant melanomas. Also used in the analysis were MAbs against human VEGFR-2 (Sigma, St. 15Louis, MO) and polyclonal antibodies against VEGFR-3 (affinity purified anti-human Flt-4 antibodies; R & D Systems, Minneapolis, MN). A MAb raised to the receptor for granulocyte colony stimulating factor, designated LMM774 (Layton et al., Growth Factors 14: 117-130, 1997), was used as a negative control. Like 20the VEGF-D MAbs, LMM774 was of the mouse IgG 1 isotype and therefore served as an isotype-matched control antibody. Five micrometer thick sections from formalin fixed and paraffin embedded tissue of the cutaneous malignant melanomas were used as the test tissue. The sections were dewaxed and rehydrated and 25then washed with PBS. The primary antibodies were incubated with tissue sections at concentrations of 5-40 pg/ml depending on incubation time. Step omission controls, in which primary antibodies were omitted, were carried out in parallel as were adsorption controls in which anti-VEGF-D MAbs were incubated with 30a 40-fold molar excess of VEGF-DANAC for 1 hour at room temperature prior to incubation with tissue sections. Isotype matched controls with the LMM774 antibody were also carried out. - 36 - Detection of alkaline phosphatase-conjugated secondary antibody was achieved using Fast Red Substrate (Sigma, St. Louis, MO). In some cases, tissue sections were bleached of melanin prior to immunohistochemistry by incubation in 0.25% potassium 5permanganate for 3 hours followed by a six minute incubation in 1% oxalic acid. In these cases, detection of peroxidase conjugated secondary antibody was with 3,3'-diaminobenzadine (DAB) (Dako Corp., Carpinteria, CA). Positive reactions were seen with all three VEGF-D MAbs with essentially the same staining patterns. VEGF-D immunoreactivity was detected in 13 of the 15 melanomas tested. The melanomas showed patterns of reaction ranging from homogeneous staining throughout the lesion to localization of the reaction at the invasive periphery of the lesion. 15. Figures 5A-5H show the results of immunohistochemical analysis from two tumors exemplifying the different reaction patterns. Antibody detection in Figures 5A and 5B was with Fast Red Substrate (red color denotes positive signal), and in Figures 5C-5H was with DAB (brown color denotes positive signal). The 20tissue sections shown in Figures 5C-5H were bleached of melanin prior to incubation with antibody. The VEGF-D antibody used in all panels except Figures 5E and 5G was VD1 (4A5) . Scale bars in Figure 5A denote 150pm, in Figures 5B-5D 20pm and in Figures SE-5H 10pm. 25 As seen in Figures 5A and SB, heterogeneous staining was apparent through the bulk of the first melanoma. In this tumor, the detected VEGF-D staining is more pronounced in the intradermal nests of tumor cells (white arrowheads) at the periphery of the invasive portions of the main bulk of the tumor, 30and is less intense or undetectable in the central portion. VEGF-D is also detected in small capillary-sized vessels (white arrows) in the papillary and reticular dermis adjacent to - 37 positive reacting tumor cells (Figure 5B) and in thicker-walled blood vessels of pre-capillary and post-capillary venule size. As seen in Figure SC, in the second melanoma, VEFG-D is more evenly distributed throughout the tumor mass and was detected in Vessels in the tumor as well as in tumor cells. Regions of stroma which stained negative are denoted by black asterisks. For both of the above-mentioned tumors, upper dermal capillary vessels and other blood vessels at a distance from the tumor, and in the mid and deep reticular dermis away from the 10tumor and sweat glands, showed very weak or no vessel wall staining and did not exhibit the granular cytoplasmic endothelial cell staining seen in the small vessels adjacent to the immunoreactive tumor cells. Non-neoplastic junctional melanocytes were also negative indicating that VEGF-D is not 15expressed by this cell type in adult skin. Figure 5D, which is a serial section control for the tissue of Figure 5C, shows that the adsorption control was negative. Step omission and isotype matched controls were also negative. Sections of malignant melanoma were analyzed for 20localization of VEGFR-3, a receptor for VEGF-D which is expressed on the endothelial cells of lymphatic vessels in adult tissues (Lymboussaki, A. et al., Am. J. Pathol. 153: 395-403, 1998) . As seen in Figure 5E, VEGFR-3 was detected in the endothelial cells of a thin-walled vessel (white arrow) in the melanoma. The 25VEGFR-3 positive vessels adjacent to tumor cells were also positive for VEGF-D (Figure 5F), as assessed by immunohistochemical analysis of serial sections, indicating that the VEGF-D immunoreactivity in these vessels 'may arise due to receptor-mediated uptake into endothelial cells. Sections were 30also analyzed by immunohistochemistry for localization of VEGFR 2. VEGFR-2 is known to be upregulated in the endothelium of blood vessels in tumors (Plate, K. et al., Cancer Res, 53: 5822 - 38 - 5827, 1993) . As seen in Figure 5G, VEGFR-2 was detected in the endothelium of blood vessels (white arrow) and in the nearby melanoma. Some of the vessels that were immunopositive for VEGFR-2 were also positive for VEGF-D (white arrow in Figure 5H) Indicating that VEGF-D uptake into tumor vessels could be mediated by this receptor also. Example 5 VEGF-D in lung cancer Neoangiogenesis is thought to be a useful prognostic 10indicator for non-small cell lung carcinoma (NSCLC) (Fontanini, G. et al., Clin Cancer Res. 3: 861-865, 1997). Therefore localization of VEGF-D was analyzed in a case of squamous cell carcinoma of the lung by immunohistochemistry (Figures 6A-6F) . The immunohistochemistry was conducted as in Example 4, except 15that antibodies to alpha-smooth muscle actin (DAKO Corp., Carpinteria, CA) were also used to immunostain. The anti-VEGF-D MAb used for immunostaining in Figures 6A and 6D was VD1 (4A5). Figure 6A shows that VEGF-D is detected in tumor cells that form an island at the center of the photomicrograph, in cells lining 20the adjacent large vessel and in cells within the desmoplastic stroma. The desmoplastic stroma is indicated by a black bracket and the dotted box denotes the region shown in higher power in Figure 6D. The immunopositive cells in the stroma may be myofibroblasts. 25 - Figure 6B shows that VEGFR-2 is detected in cells lining the large vessel. However, these vessels were negative for VEGFR-3 in this tumor. The dotted box denotes the region shown in higher power in Figure 6E. Control staining, of a tissue section from the same case, in which VEGF-D MAb had been preincubated with a 3040-fold molar excess of the VHD of human VEGF-D gave no signal (Figure 6C). - 39 - As mentioned above, the immunopositive cells in the desmoplatic stroma may be myofibroblasts. Therefore, the desmoplastic stroma was immunostained using MAbs specific for alpha-smooth muscle actin that detect myofibroblasts. As seen 5in Figure 6F, the stroma stained positive, indicating the presence of myofibroblasts. Secretion of an angiogenic factor by stromal components may serve to amplify the angiogenic stimulus generated by the tumor. lOExample 6 VEGF-D in breast cancer Localization of VEGF-D was also analyzed in breast ductual carcinoma in situ by immunohistochemistry, the results of which are shown in Figures 7A-7F. The immunohistochemistry was conducted as in Example 4, except MAbs specific for alpha-smooth 15muscle actin (DAKO Corp., Carpinteria, CA) and the platelet/endothelial adhesion molecule (PECAM) (DAKO Corp., Carpinteria, CA) were also used to immunostain. The anti-VEGF-D MAb used for immunostaining in Figure 7A was VD1 (4A5). As seen in Figure 7A, VEGF-D was detected in tumor cells in 20the ducts and in small so-called "necklace" vessels (denoted by black arrowheads) immediately adjacent to the basal lamina of the tumor-filled ducts. The necklace vessels were also positive for VEGFR-2 (Figure 7C), VEGFR-3 (Figure 7D) and PECAM (Figure 7E) as indicated by the black arrowheads. PECAM is a classic marker 25for endothelium and is also found on platelets and leukocytes. PECAM plays a role in the emigration of leukocytes to inflammatory sites (Muller et al., J. Exp. Med. 178: 449-460). PECAM antibody staining on the "necklace" vessels helps to confirm that these structures are vessels. The edge of the duct 30is identified by staining for alpha-smooth muscle actin (Figure 7B) that detects myofibroblasts. Control staining, of a tissue section serial to that shown in Figure 7A, in which VEGF-D MAb - 40 had been preincubated with a 40-fold molar excess of the VHD of human VEGF-D gave no signal (Figure 7F) . These findings indicate that VEGF-D, secreted by the tumor cells, could activate its receptors on vessels in the vicinity and thereby play a role in 5attracting the growth of the necklace vessels to their positions very close to the ducts. This could be of importance both for solid tumor growth and metastatic spread. Example 7 VEGF-D in endometrial cancer 10 VEGF-D was also detected in endometrial adenocarcinoma (Figure 8) . The immunohistochemistry was carried out as in Example 4 using the anti-VEGF-D MAb VD1 (4A5). Moderate staining for VEGF-D was seen in the glandular tumor cells (GL), very strong reactivity was seen in the myofibroblastic cells of 15the desmoplastic stroma (DM) at the advancing invasive edge of the tumor and strong reactivity in the endothelium and walls of adjacent blood vessels (black arrows) in the myometrium (Myo). Interestingly, VEGF-D reactivity was particularly strong in the myofibroblasts of the desmoplastic stroma, indicating that the 20glandular tumor cells can induce VEGF-D expression in these fibroblasts which would amplify the angiogenic potential of the tumor. As expression of VEGF-D in cells of the desmoplastic stroma was also detected in lung carcinoma (Figure 6A), it may be that a range of tumors can induce VEGF-D in stromal 25components. This is analogous to the developing lung where the mesenchymal cells, presumably fibroblastic precursors, strongly express the VEGF-D gene. Therefore, signals from both embryonic and tumor tissues can induce expression of VEGF-D in fibroblasts. 30Example 8 VEGF-D in non-tumorigenic tissue. . Tissues with a high cell turn-over and/or metabolic load, such as the colon, require an extensive vascular network. - 41 - Therefore the human colon was analyzed for localization of VEGF-D by immunohistochemistry, the results of which are shown in Figures 9A-9F. The immunohistochemistry was conducted as in Example 4, except that antibodies specific for alpha-smooth 5muscle actin (DAKO Corp., Carpinteria, CA) were also used to immunostain. For all tissue sections shown, detection was with DAB (brown color denotes positive signal) and for Figures 9A, 9B, 9C and 9F, the VEGF-D antibody used was VD1 (4A5) . For clarity, counterstaining was omitted in Figures 9A, 9B, 9D and 9F. The 10scale bar in Figure 9A denotes 120 pm, in Figures 9B, 9D and 9F denotes 40 pm and in Figures 9C and 9E denotes 6 pm. VEGF-D was localized in blood vessels of the submucosa (Figure 9A). Higher power analysis reveals staining of vascular smooth muscle (white arrowheads), but not of the endothelial l5cells (black arrowheads) in arterioles (Figures 9B and 9C). Staining of a serial section to that shown in Figures 9A-9C with antibody specific for alpha-smooth muscle actin detects vascular smooth muscle (white arrowheads) but not the endothelium (black arrowheads) (Figures 9D and 9E). This staining demonstrates that 20the VEGF-D reactivity was in vascular smooth muscle cells of arterioles. Furthermore, these endothelial cells did not exhibit immunoreactivity for either VEGFR-2 or VEGFR-3, indicating that these cells cannot accumulate VEGF-D in a receptor-mediated fashion. Preincubation of the VEGF-D MAb with a 40-fold molar 25excess of the VHD of human VEGF-D completely blocks the staining of vascular smooth muscle (Figure 9F). As the colon is subject to a variety of insults, some of which cause vascular damage, VEGF-D in the submucosa may be produced by vascular smooth muscle cells in preparation for 30vascular regeneration. Upon activation of the endothelium in response to vascular damage, up-regulation of VEGFR-2 on endothelial cells of these vessels would allow the VEGF-D, - 42 produced by the vascular smooth muscle, to induce endothelial cell proliferation and vessel repair. Up-regulation of VEGFR-2 by the endothelium of small arterioles and microvessels in response to arterial damage has been reported previously in the 5context of ischemic stroke (Issa, R. et al., Lab Invest 79: 417 425, 1999). Example 9 Role of VEGF-D in Tumor Development In order to generate cell lines constitutively over 10expressing derivatives of VEGF-D, regions of the human VEGF-D cDNA were inserted into the mammalian expression vector Apex-3 (Evans et al, Mol. Immunol., 1995 32 1183-1195) . This vector is maintained episomally when transfected into 293-EBNA human embryonal kidney cells. For expression of mature VEGF-D, the 15region of pEFBOSVEGF-DANAC containing the sequences encoding the IL-3 signal sequence, the FLAG* octapeptide and the mature VEGF-D were inserted into the XbaI site of Apex-3 (see Example 9 in International Patent Application PCT/US97/14696 (W098/07832) ) . The resulting plasmid was designated pVDApexDANAC (Stacker, S.A. 20et al., J Biol Chem 274: 32127-32136, 1999 and see Example 1 in International Patent Application PCT/US98/27373) . The entire disclosure of the International Patent Application PCT/US98/27373 is incorporated herein by reference. A similar construct was made for expression of the unprocessed full-length VEGF-D tagged 25at the N-terminus with Flag*. In this construct, the DNA encoding the VEGF-D signal sequence for protein secretion was deleted and substituted with DNA encoding the IL-3 signal sequence, followed by the FLAG* octapeptide and two amino acids (Thr-Arg) immediately upstream and in the same reading frame as 30DNA encoding residues 24-354 of VEGF-D. This construct was designated pVDApexFull-N-Flag (Stacker, S.A. et al., J Biol Chem 274: 32127-32136, 1999 and see Example 1 in International Patent - 43 - Application PCT/US98/27373) . These vectors were transfected into cells of the human embryo kidney cell line 293EBNA-1 by the calcium phosphate method or with Fugene* according to the manufacturer's instructions (Roche Molecular Biochemicals, 5Mannhiem, Germany), and stable transfectants were selected in the presence of 100 pg/ml hygromycin supplemented DMEM. Cell lines expressing high levels of VEGF-D-Full-N- Flag and VEGF-D8NAC were subsequently identified by metabolic labeling, immunoprecipitation and Western blot analysis (Stacker, S.A. et 10al., J Biol Chem 274: 32127-32136, 1999 and see Example 1 in International Patent Application PCT/US98/27373). Six to eight weeks old SCID mice (ARC, Perth, Australia) were injected subcutaneously in the mammary fat pad with 2 x 107 of the transfected 293 cells or untransfected parental 293 cells 15in PBS. Tumors were allowed to grow and were measured with digital calipers over a period of three weeks. Experiments were terminated after three weeks when the first animal reached the maximum size allowed by the Institutional Ethics Committee. The tumor size was calculated as the width x length x 0.6 x (width 20x length)/2. Figure 10 shows the results of the analysis of tumors in SCID mice resulting from injection of untransfected parental 293 cells (designated "293") or 293 cells transfected with the construct encoding VEGF-D-FULL-N-FLAG (designated "VEGF-D-293") . 25There is significant difference between the tumors derived from the 293-VEGF-D-FULL-N-FLAG cells and those derived from the untransfected 293 cells. After three weeks the mean tumor size of the 293-VEGF-D-FULL-N-FLAG group was 937± 555 mm 3 (mean ± SD, n = 8) compared to 136± 230 mm 3 for the untransfected 293 cells 30(n = 8). Interestingly, tumors generated from 293 cells transfected with a construct encoding VEGF-DANAC were not - 44 significantly different in size, 50 + 76 mm 3 (n = 7), to those from the untransfected 293 cells. In addition, the macroscopic appearance of tumors derived from the untransfected 293 cells was one of a pale white surface, compared to the tumors derived from the 293-VEGF-D-FULL-N-FLAG cells which had a bloody appearance, with the presence of blood vessels apparent throughout the tumor. Also, sections were analyzed by immunohistochemistry with an anti-PECAM monoclonal antibody (Pharmingen, San Diego, CA), 10a marker of endothelial cells. Sections of tumors generated with 293-VEGF-D-FULL-N-FLAG cells demonstrated a marked increase in PECAM expression compared to the tumors generated with untransfected parental 293 cells. This analysis confirms the much greater abundance of blood vessels in the tumors expressing 15unprocessed full-length VEGF-D. This experiment indicates that the unprocessed form of VEGF D is capable of inducing tumor angiogenesis and the growth of a solid tumor in vivo. Interestingly, the tumors derived from cells expressing the mature, fully processed form of VEGF-D 20showed no increase in growth compared to the untransfected 293 parental cells. This indicates the importance of the propeptides (N-pro and C-pro) in VEGF-D for the correct localization or function of the VHD of VEGF-D. An explanation for this result is that the propeptides are involved in matrix association and 25only when VEGF-D is positioned correctly on the extracellular matrix or cell surface heparin sulphate proteoglycans is the growth factor able to induce angiogenesis and/or lymphangiogenesis. An alternative explanation is that the propeptides increase the half-life of the VEGF-D VHD in vivo. 30 - 45 - Example 10 VEGF-D induction of tumor angiogenesis To determine whether VEGF-D plays a role in tumor angiogenesis, 293EBNA cell lines expressing VEGF or VEGF-D were generated. 293EBNA cells normally do not express detectable Levels of VEGF, VEGF-C, or VEGF-D, the ligands that activate VEGFR-2 and/or VEGFR-3 (Stacker, S.A., et al. , Growth Factors 17: 1-11 (1999) ) . 293EBNA cells produce slow growing and poorly vascularized epithelioid-like tumors in immunodeficient mice. Western-blot analysis of conditioned medium from the generated 10293EBNA cell lines in vitro showed that the mature forms of the active growth factors were secreted. Six to twenty-one week old female SCID or SCID/nod mice (Animal Resources Center, Canning Vale, Australia; Austin Research Institute, Australia; and Walter and Eliza Hall 15Institute for Medical Research, Australia) were placed in groups of 6 to 10 mice and injected subcutaneously in the mammary fat pad with cell lines expressing VEGF-293, VEGF-D-293, or control 293 cell lines at a concentration of 2.0-2.5 x 107 in culture medium. Tumor growth and morphology were analyzed over 35 days. 20Tumors were measured with digital calipers and tumor volume was calculated by the formula: volume = length x width 2 x 0.52. Three to five weeks after injection with cell lines the mice were euthanized and the tumors were removed for examination. VEGF-D 293 tumors and 293 tumors were excised post mortem on day 25 and 25weighed. VEGF-293 cells produced tumors with an increased growth rate compared with control 293 cells. The VEGF-293 tumors were highly vascularized with extensive edema, consistent with VEGF being a potent tumor angiogenesis factor and an inducer of vascular 30permeability. VEGF-D-293 cells also showed enhanced growth in vivo and the tumors were highly vascularized compared with - 46 control 293 tumors but showed no evidence, overtly or microscopically, of edema. Tumor growth arising from injection of VEGF-D-293 cells was blocked by twice weekly intraperitoneal injections of monoclonal antibody VD1, an antibody specific for the bioactive region of VEGF-D that blocks binding of VEGF-D to VEGFR-2 and VEGFR-3. However, tumor growth was unaffected by treatment with a control, isotype-matched antibody. Treatment with the VD1 antibody reduced the abundance of 10vessels in the tumors as assessed by immunohistochemistry for the endothelial cell marker PECAM-1. Western blotting demonstrated the expression of VEGF-D and VEGF in VEGF-D-293 and VEGF-293 tumors, respectively, and also that VEGF was not upregulated in VEGF-D-293 tumors. Analysis of tumor weights post 15mortem demonstrated a significant difference- between the VEGF-D 293 tumors (0.49±0.22 g, n=7; mean ± SD) and the control 293 tumors (0.123±0.118 g, n=9, p=0.01). Example 11 VEGF-D induction of tumor lymphangiogeriesis 20 Because metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors, experiments were conducted to determine if VEGF-D induced tumor lymphangiogenesis, or if expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes. 25 To analyze the role of VEGF-D in tumor spread, VEGF-D-293 tumors were induced in SCID/NOD mice (Animal Resources Center, Canning Vale, Australia; Austin Research Institute, Australia; and Walter and Eliza Hall Institute for Medical Research, Australia) . Post-mortem analysis revealed that animals with 30VEGF-D-293 tumors had developed- metastatic lesions in either the lateral axillary lymph node and/or superficial inguinal nodes in 14 of 23 animals compared with 0 of 16 animals for VEGF-293 - 47tumors and 0 of 14 animals for 293 tumors. In some cases, the spread of metastatic tumor cells from the primary tumor in SCID/NOD mice was evident as a trail of tumor cells in the lymphatics of the skin between the primary tumors and the lateral 5axillary node. Treatment of mice harboring VEGF-D-293 tumors with the VDl monoclonal antibody (Table 1) blocked the metastatic spread to lymph nodes. None of the 7 mice treated over 25 days with VD1 exhibited lymphatic spread, whereas 6 of 10 mice treated with a 10control isotype-matched monoclonal antibody exhibited lymphatic spread. These results indicate that VEGF-D can promote the metastatic spread of these tumors via the lymphatics. Table 1: Metastatic spread of tumors in SCID/NOC mice 15 Tumor line Number of mice with Number of mice with primary tumors spread to local lymph nodes VEGF-D-293 23 14(61%) VEGF-D-293 7 0 (VD1-treated) a VEGF-D-293 10 6(60%) 20 (LMM774-treated)b a Purified monoclonal antibodies were injected twice weekly over the course of the experiment, starting 1 day after injection of the tumor cells. VD1 is a neutralizing monoclonal antibody 25against VEGF-D. b LMM774 is an isotype-matched control monoclonal antibody that does not bind VEGF-D. The data show that expression of VEGF-D can promote 30metastatic spread of tumor cells through the lymphatic network. - 48 - VEGF-D induced formation of lymphatic vessels in the tumors, as detected by immunohistochemistry for the lymphatic-specific marker LYVE-1, presumably through the lymphatic receptor VEGFR-3, although activation of VEGFR-3-VEGFR-2-heterodimers cannot be excluded. The expression of lymphangiogenic factors alone is sufficient to induce the formation of lymphatic vessels in the center of a tumor and to facilitate the metastatic spread to the lymph nodes. VEGF-D was localized to tumor cells and the endothelium of lOvessels in malignant melanoma, lung and breast cancers (see Examples 4-6). Example 12 Variance in tumor characteristics induced by different forms of VEGF-D 15 In addition to the determination of the role of VEGF-D in tumor angiogenesis and lymphangiogenesis, the methods of Example 10 and 11 were used to produce and evaluate tumors expressing different forms of VEGF-D which represent the cleavage of the N, C, and both N and C terminal propeptides. The cell lines 20injected into the mice were 293EBNA, VEGF-D-293, VEGF-DANAC-293, VEGF-DAC-293 (cells expressing VEGF-D lacking the C-terminal propeptide), and VEGF-DAN-293 (cells expressing VEGF-D lacking the N-terminal propeptide). The tumors produced by the VEGF-DAN cells grew more rapidly 25than the tumors produced by control cells. Upon morphological examination the tumors were red in appearance and contained a significant vascular reaction, including a substantial fluid component not seen in the control tumors. The tumors produced by the VEGF-DAN cells had significant differences in growth and 30morphological characteristics than the control tumors. The graph of Figure 11 shows the increased rate of growth in tumors from the VEGF-DAN cells. The tendency toward fluid - 49 accumulation in the tumors produced by the VEGF-DAN cells can be seen in Figure 12, a photograph of such a tumor. This can be contrasted with the photograph of Figure 13 which depicts a normal tumor such as that produced by the control cells. 5 The tumors produced by the VEGF-DAC cells grew in a similar fashion to the control cells and did not exhibit excess fluid formation. The tumors produced by the VEGF-DANaC cells grew very slowly compared to the control tumors. The VEGF-DANAC tumors formed in 10about 70 days as compared to an average 30-35 days for the control tumors and 20-25 days for the VEGF-DAN tumors. Examination of these tumors showed that they had a reduced vascular response, having fewer blood vessels than control tumors by PECAM-1 staining. The tumors developed lymphatic networks as l5shown by LYVE-1 staining and induced formation of lymphatic metastases. The graph of Figure 14 shows the decreased rate of growth in tumors from the VEGF-DANAC cells. The localization of VEGF-D in malignant melanoma is consistent with a role for this molecule in tumor angiogenesis 20as strong signals for VEGF-D were detected in the endothelial cells of blood vessels near immunopositive tumor cells, but not in vessels distant from tumor cells. This indicates that VEGF-D found on vessels in or near the tumor may arise due to receptor mediated uptake, which supports the hypothesis that VEGF-D, 25secreted by tumor cells, binds and accumulates in target endothelial cells thereby establishing a paracrine mechanism regulating tumor angiogenesis. A similar pattern of VEGF localization in tumor cells and tumor blood vessels was reported previously (Plate, K. et al., Brain Pathology 4: 207-218, 1994) . 30Consistent with the hypothesis that VEGF-D plays a role in tumor angiogenesis is the finding that a receptor for VEGF-D, VEGFR-2, is upregulated in 'the endothelial cells of blood vessels in - 50 tumors (Plate, K. et al., Cancer Res 53: 5822-5827, 1993). Indeed, some of the VEGF-D immunopositive vessels detected in the melanomas studied here were also positive for VEGFR-2. Signaling via VEGFR-2 is critical for sustaining tumor angiogenesis 5(Millauer, B. et al., Cancer Res 56: 1615-1620, 1996) and the angiogenic activity of VEGF-D in vivo (Marconcini, L. et al., Proc Natl Acad Sci USA 96: 9671-9676, 1999) is most likely mediated by this receptor. Similar patterns of staining to those seen in the melanomas were observed in squamous cell carcinoma 10of the lung and breast ductal carcinoma in situ (BDCIS) as VEGF-D was detected in tumor cells and on vessels nearby. Vessels near the tumor-filled ducts in BDCIS and near the islands of tumor cells in lung carcinoma were also positive for VEGFR-2, again suggesting this ligand and receptor may contribute to the control 15of tumor angiogenesis in a paracrine fashion. These results also indicate that VEGF-D may play a role in stimulating the growth of lymphatic vessels in the vicinity of malignant melanoma as vessels positive for VEGFR-3, a receptor for VEGF-D expressed on lymphatic endothelium in normal adult 20tissues, were also positive for VEGF-D. Similar staining patterns were seen in BDCIS as some of the VEGF-D positive vessels surrounding the tumor-filled ducts were also positive for VEGFR-3. Signaling via VEGFR-3 is thought to be important for lymphangiogenesis (Taipale, J. et al., Curr Top Microbiol Immunol 25237: 85-96, 1999), although this receptor can be up-regulated on blood vessel capillaries in cancer (Valtola, R. et al., Am. J. Path. 154: 1381-1390, 1999). Therefore the paracrine regulatory system consisting of VEGF-D and VEGFR-3 could stimulate both lymphangiogenesis and angiogenesis in cancer. Accordingly, the 30route by which a tumor metastasizes may be determined, in part, by its capacity to induce angiogenesis and/or lymphangiogenesis. If so, the expression by tumor cells of soluble growth factors - 51 which are purely angiogenic (e.g. VEGF) as opposed to those which may also induce lymphangiogenesis (e.g. VEGF-D) could be an important determinant of the route of metastatic spread. VEGF-D may also play a role in vascular maintenance in non .5tumorigenic tissues. In the arterioles of the submucosa of the colon, VEGF-D was localized in vascular smooth muiscle, not in the endothelium. The absence of VEGF-D in the endothelium is probably a consequence of the lack of expression of the VEGF-D receptors VEGFR-2 and VEGFR-3 in endothelial cells. Activation 10of the endothelium in response to vascular damage is probably sufficient to induce expression of VEGFR-2 by endothelial cells (Issa, R. et al., Lab. Invest. 79: 417-425, 1999) which would, in turn, render the VEGF-D, produced by vascular smooth muscle, capable of inducing endothelial cell proliferation and thus 15affec'ting vessel repair. The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur 20to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof. The entire disclosure in the complete specification of our Australian Patent Application No. 41946/01 is by this cross-reference incorporated into the present specification. - 52 -
Claims (13)
1. A method of diagnosing growth characteristics of a neoplastic disease in an organism, the method comprising: 5 (a) measuring amount of unprocessed VEGF-D polypeptide in a sample from an organism with a neoplastic disease; and (b) diagnosing growth characteristics of the neoplastic disease from the amount of the VEGF-D measured 10 in step (a), wherein increased unprocessed VEGF-D in said sample correlates with increased tumor growth or metastatic risk.
2. A method of diagnosing growth characteristics of a 15 tumor in an organism, the method comprising: (a) measuring amount of unprocessed VEGF-D polypeptide in a sample from an organism with a neoplastic disease; and (b) diagnosing growth characteristics of the tumor 20 from the amount of the VEGF-D measured in step (a), wherein increased unprocessed VEGF-D in said sample correlates with increased tumor growth or metastatic risk.
3. The method according to claim 1 or claim 2, wherein 25 the measuring step comprises exposing said sample to a composition comprising an antibody that specifically binds VEGF-D.
4. The method according to claim 3, wherein said 30 antibody is a monoclonal antibody.
5. The method according to claim 3 or claim 4, wherein said antibody includes a detectable label. 35
6. The method according to any one of the preceding claims, wherein said unprocessed VEGF-D has an apparent 53 N:\lborme\Cae\Patnt\46004999\P46490.jAU.t\spcci\4~449.AU.1 amendments 2009-3-33.doc molecular weight of -53K.
7. The method according to any one of the preceding claims, wherein said sample is selected from the group 5 consisting of tissue, blood, serum, plasma, urine, ascities fluid and pleural effusion.
8. The method according to claim 7, wherein said sample is a tissue sample. 10
9. The method according to claim 1 or claim 2, wherein said sample comprises endothelial cells.
10. The method according to claim 1 or claim 2, wherein 15 said sample comprises a lymph node.
11. The method according to any one of the preceding claims, wherein said neoplastic disease is selected from the group consisting of malignant melanoma, breast ductal 20 carcinoma, squamous cell carcinoma, prostate cancer and endometrial cancer.
12. A method according to any preceding claim, wherein the organism is a human. 25
13. A method according to any preceding claim, substantially as hereinbefore described, with reference to the examples and/or figures. 54 N.\MelhOUrne\CaeS\Patent\46000-46999\P46490.AU.3\Spec g\P46490-AU.1 amendments 2009-3-23.doc
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2006201128A AU2006201128B2 (en) | 2000-03-02 | 2006-03-17 | Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60186361 | 2000-03-02 | ||
AU41946/01A AU4194601A (en) | 2000-03-02 | 2001-03-02 | Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor |
AU2006201128A AU2006201128B2 (en) | 2000-03-02 | 2006-03-17 | Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU41946/01A Division AU4194601A (en) | 2000-03-02 | 2001-03-02 | Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor |
Publications (2)
Publication Number | Publication Date |
---|---|
AU2006201128A1 AU2006201128A1 (en) | 2006-04-13 |
AU2006201128B2 true AU2006201128B2 (en) | 2009-05-28 |
Family
ID=36251973
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2006201128A Ceased AU2006201128B2 (en) | 2000-03-02 | 2006-03-17 | Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D |
Country Status (1)
Country | Link |
---|---|
AU (1) | AU2006201128B2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102007038730A1 (en) * | 2007-08-16 | 2009-02-19 | Carl Zeiss Meditec Ag | Evidence of Human Vascular Endothelial Growth Factor |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998007832A1 (en) * | 1996-08-23 | 1998-02-26 | Ludwig Institute For Cancer Research | Recombinant vascular endothelial cell growth factor d (vegf-d) |
WO1999033485A1 (en) * | 1997-12-24 | 1999-07-08 | Ludwig Institute For Cancer Research | Expression vectors and cell lines expressing vascular endothelial growth factor d, and method of treating melanomas |
EP0935001A1 (en) * | 1996-07-15 | 1999-08-11 | Chugai Research Institute for Molecular Medicine Inc. | Novel vegf-like factors |
-
2006
- 2006-03-17 AU AU2006201128A patent/AU2006201128B2/en not_active Ceased
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0935001A1 (en) * | 1996-07-15 | 1999-08-11 | Chugai Research Institute for Molecular Medicine Inc. | Novel vegf-like factors |
WO1998007832A1 (en) * | 1996-08-23 | 1998-02-26 | Ludwig Institute For Cancer Research | Recombinant vascular endothelial cell growth factor d (vegf-d) |
WO1999033485A1 (en) * | 1997-12-24 | 1999-07-08 | Ludwig Institute For Cancer Research | Expression vectors and cell lines expressing vascular endothelial growth factor d, and method of treating melanomas |
Also Published As
Publication number | Publication date |
---|---|
AU2006201128A1 (en) | 2006-04-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20010038842A1 (en) | Methods for treating various cancers expressing vascular endothelial growth factor D, for screening for a neoplastic disease and for maintaining vascularization of tissue | |
US7534572B2 (en) | Methods for treating neoplastic disease characterized by vascular endothelial growth factor D expression, for screening for neoplastic disease or metastatic risk, and for maintaining vascularization of tissue | |
EP1054687B1 (en) | Expression vectors and cell lines expressing vascular endothelial growth factor d, and method of treating melanomas | |
EP1140175B1 (en) | Antibodies to truncated vegf-d and uses thereof | |
Nikol et al. | Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions. | |
AU2001264565B2 (en) | A method for activating only the vascular endothelial growth factor receptor-3 and uses thereof | |
US20060198844A1 (en) | Bone morphogenetic protein-2 in the treatment and diagnosis of cancer | |
AU2001264565A1 (en) | A method for activating only the vascular endothelial growth factor receptor-3 and uses thereof | |
EP1519193B1 (en) | Methods for detecting cancers expressing vascular endothelial growth factor D | |
AU2006201128B2 (en) | Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D | |
US20050250103A1 (en) | Methods for detecting for the presence of tumor cells and for screening for anti-tumor agents | |
AU765888B2 (en) | Expression vectors and cell lines expressing vascular endothelial growth factor D, and method of treating melanomas | |
AU2007200999A1 (en) | Methods for detecting for the presence of tumor cells and for screening for anti-tumor agents | |
Li | The role of CD105 and its ligand transforming growth factor β in angiogenesis | |
Partanen | Lymphatic vs blood vascular endothelial growth factors and receptors in human tissues and diseases | |
US20050209136A1 (en) | Method for stimulating connective tissue growth or wound healing |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PC1 | Assignment before grant (sect. 113) |
Owner name: VEGENICS LIMITED Free format text: FORMER APPLICANT(S): LUDWIG INSTITUTE FOR CANCER RESEARCH |
|
MK4 | Application lapsed section 142(2)(d) - no continuation fee paid for the application | ||
NA | Applications received for extensions of time, section 223 |
Free format text: AN APPLICATION TO EXTEND THE TIME FROM 02 MAR 2008 TO 02 OCT 2008 IN WHICH TO PAY THE CONTINUATION FEE HAS BEEN FILED . |
|
NB | Applications allowed - extensions of time section 223(2) |
Free format text: THE TIME IN WHICH TO PAY THE CONTINUATION FEE HAS BEEN EXTENDED TO 02 OCT 2008. |
|
FGA | Letters patent sealed or granted (standard patent) | ||
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |