CA2310252A1 - Active immunization against angiogenesis-associated antigens - Google Patents
Active immunization against angiogenesis-associated antigens Download PDFInfo
- Publication number
- CA2310252A1 CA2310252A1 CA002310252A CA2310252A CA2310252A1 CA 2310252 A1 CA2310252 A1 CA 2310252A1 CA 002310252 A CA002310252 A CA 002310252A CA 2310252 A CA2310252 A CA 2310252A CA 2310252 A1 CA2310252 A1 CA 2310252A1
- Authority
- CA
- Canada
- Prior art keywords
- immunogen
- molecule
- mammal
- antibody
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 55
- 102000036639 antigens Human genes 0.000 title claims abstract description 55
- 108091007433 antigens Proteins 0.000 title claims abstract description 55
- 230000033115 angiogenesis Effects 0.000 title claims description 56
- 230000003053 immunization Effects 0.000 title description 3
- 238000002649 immunization Methods 0.000 title description 3
- 238000000034 method Methods 0.000 claims abstract description 80
- 230000002491 angiogenic effect Effects 0.000 claims abstract description 36
- 230000002163 immunogen Effects 0.000 claims description 63
- 241000124008 Mammalia Species 0.000 claims description 41
- 210000004027 cell Anatomy 0.000 claims description 35
- 239000012634 fragment Substances 0.000 claims description 21
- 230000028993 immune response Effects 0.000 claims description 21
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 19
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 18
- 210000004443 dendritic cell Anatomy 0.000 claims description 17
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 claims description 16
- 239000013598 vector Substances 0.000 claims description 15
- 239000002671 adjuvant Substances 0.000 claims description 12
- 210000000612 antigen-presenting cell Anatomy 0.000 claims description 12
- 108020004707 nucleic acids Proteins 0.000 claims description 12
- 102000039446 nucleic acids Human genes 0.000 claims description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 12
- 229920001184 polypeptide Polymers 0.000 claims description 10
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 102000006495 integrins Human genes 0.000 claims description 9
- 108010044426 integrins Proteins 0.000 claims description 9
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 8
- 150000003384 small molecules Chemical class 0.000 claims description 6
- 230000004614 tumor growth Effects 0.000 claims description 6
- 206010003246 arthritis Diseases 0.000 claims description 5
- 102000043131 MHC class II family Human genes 0.000 claims description 4
- 108091054438 MHC class II family Proteins 0.000 claims description 4
- 241001529936 Murinae Species 0.000 claims description 4
- 101100481403 Bos taurus TIE1 gene Proteins 0.000 claims description 3
- 102000043129 MHC class I family Human genes 0.000 claims description 3
- 108091054437 MHC class I family Proteins 0.000 claims description 3
- 201000004681 Psoriasis Diseases 0.000 claims description 3
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 210000004408 hybridoma Anatomy 0.000 claims description 3
- 208000002780 macular degeneration Diseases 0.000 claims description 3
- 108010031318 Vitronectin Proteins 0.000 claims description 2
- 102100035140 Vitronectin Human genes 0.000 claims description 2
- 108010018828 cadherin 5 Proteins 0.000 claims description 2
- 230000005847 immunogenicity Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 9
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 claims 5
- 102100029761 Cadherin-5 Human genes 0.000 claims 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims 1
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 claims 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 claims 1
- 230000003278 mimic effect Effects 0.000 abstract description 4
- 230000000890 antigenic effect Effects 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 31
- 102000004169 proteins and genes Human genes 0.000 description 20
- 235000018102 proteins Nutrition 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 18
- 102000005962 receptors Human genes 0.000 description 18
- 108020003175 receptors Proteins 0.000 description 18
- 206010028980 Neoplasm Diseases 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 12
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 230000003302 anti-idiotype Effects 0.000 description 9
- 210000002889 endothelial cell Anatomy 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000000816 peptidomimetic Substances 0.000 description 5
- 102000008076 Angiogenic Proteins Human genes 0.000 description 4
- 108010074415 Angiogenic Proteins Proteins 0.000 description 4
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 4
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 4
- 108091008605 VEGF receptors Proteins 0.000 description 4
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000021615 conjugation Effects 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 108010032595 Antibody Binding Sites Proteins 0.000 description 3
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 3
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 3
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 3
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 210000003556 vascular endothelial cell Anatomy 0.000 description 3
- 230000029663 wound healing Effects 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- 102000009088 Angiopoietin-1 Human genes 0.000 description 2
- 108010048154 Angiopoietin-1 Proteins 0.000 description 2
- 102000009075 Angiopoietin-2 Human genes 0.000 description 2
- 108010048036 Angiopoietin-2 Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 206010012689 Diabetic retinopathy Diseases 0.000 description 2
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102100022337 Integrin alpha-V Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 206010029113 Neovascularisation Diseases 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 230000005867 T cell response Effects 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 108010048673 Vitronectin Receptors Proteins 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000002870 angiogenesis inducing agent Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- 102000058223 human VEGFA Human genes 0.000 description 2
- -1 i.e. Proteins 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 102400000068 Angiostatin Human genes 0.000 description 1
- 108010079709 Angiostatins Proteins 0.000 description 1
- 108010037870 Anthranilate Synthase Proteins 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 101000645291 Bos taurus Metalloproteinase inhibitor 2 Proteins 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 229940122097 Collagenase inhibitor Drugs 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000007023 DNA restriction-modification system Effects 0.000 description 1
- 101100540419 Danio rerio kdrl gene Proteins 0.000 description 1
- 102400001047 Endostatin Human genes 0.000 description 1
- 108010079505 Endostatins Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108091008794 FGF receptors Proteins 0.000 description 1
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 1
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 1
- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 101150088608 Kdr gene Proteins 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
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000186366 Mycobacterium bovis Species 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102000004211 Platelet factor 4 Human genes 0.000 description 1
- 108090000778 Platelet factor 4 Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102100036385 Protocadherin-12 Human genes 0.000 description 1
- 101710158929 Protocadherin-12 Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 102000008790 VE-cadherin Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- JOOSFXXMIOXKAZ-UHFFFAOYSA-H [Au+3].[Au+3].[O-]C(=O)CC(S)C([O-])=O.[O-]C(=O)CC(S)C([O-])=O.[O-]C(=O)CC(S)C([O-])=O Chemical compound [Au+3].[Au+3].[O-]C(=O)CC(S)C([O-])=O.[O-]C(=O)CC(S)C([O-])=O.[O-]C(=O)CC(S)C([O-])=O JOOSFXXMIOXKAZ-UHFFFAOYSA-H 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 230000000964 angiostatic effect Effects 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000002494 anti-cea effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000008081 blood perfusion Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 208000025997 central nervous system neoplasm Diseases 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000002442 collagenase inhibitor Substances 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000005175 epidermal keratinocyte Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 201000002222 hemangioblastoma Diseases 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 125000001446 muramyl group Chemical group N[C@@H](C=O)[C@@H](O[C@@H](C(=O)*)C)[C@H](O)[C@H](O)CO 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000027758 ovulation cycle Effects 0.000 description 1
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000005747 tumor angiogenesis Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 230000004862 vasculogenesis Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical class C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464454—Enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Rheumatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Modified angiogenic self antigens and anti-idiotypic antibodies that mimic an antigenic determinant of a receptor to an angiogenic molecule are provided. In vitro and in vivo methods of using these antigens and antibodies are also provided.
Description
ACITVE IMMUNIZATION AGAINST ANGIOGENESIS-ASSOCIATED
ANTIGENS
BACKGROUND OF THE INVENTION
Current immunotherapeutic approaches to inhibiting angiogenesis rely on the passive administration of large amounts of antibodies to angiogenesis targets such as the vitronectin receptor and vascular endothelial cell growth factor (VEGF). Limitations of this therapeutic strategy include the difficulty of administering large amounts of monoclonal antibodies to a patient and maintaining a constant high level of antibodies over a long period of time in a patient.
The present invention overcomes these problems by using active specific immunotherapy against angiogenesis target molecules to inhibit angiogenesis.
Such methods of immunotherapy against angiogenic molecutes include modification of immunogens to cause an immune response against the angiogenic molecules. Modification of the target antigen can be achieved by, for example, conjugation of immunogenic reagents to the antigen (see US
Patent Nos. 5,334,379); haptenization of the antigen (see U.S. Patent Nos.
4,778,752 and 5,290,551 ); the use of adjuvants bound to, or administered with, the target antigen; binding peptide fragments to the antigen; binding the target antigen to MHC class I and class II restricted antigens (see U.S. Patent No.
4,478,823); changing giycosylation patterns of the target antigens (see U.S.
Patent No. 5,484,735); presenting the target antigen on antigen presenting cells, such as dendritic cells (see U.S. Patent Nos. 5,580,563 and 5,788,963), among other methods.
Another method where immunity is induced to an angiogenesis target molecule is that of using an anti-idiotypic antibody bearing the internal image of the target antigen. Since the mimicry of the target "self' antigen by the anti-idiotype is likely to be imperfect, such anti-idiotypes will break tolerance to a self antigen, although administration of the self antigen is not able to do so. It has been previously shown that anti-idiotypes that bear the internal image of carcinoembryonic antigen (CEA) can induce anti-CEA antibodies in patients with colorectal carcinoma, while CEA itself cannot do so. Therefore, an alternative approach to using the actual "self' angiogenesis target antigen as a vaccine is to use an anti-idiotypic antibody that mimics the antigen, bears an internal image of the antigen, and elicits an immune response.
An anti-idiotypic antibody, termed either an AB2 or an anti-idiotype, is one produced in response to the presence of an antibody in an immunologically active system. Anti-idiotypes are antibodies directed against the antigen-combining region or variable region (known as the idiotype) of another antibody molecule.
The theory of idiotypic relationships and networks is based on the Jeme model (Jeme, N.K. (1974) Ann. Immunof. {Paris) 125C: 373; Jeme, N.K. et al. (1982) EMBO 1:234). Thus immunization with an antibody expressing a paratope (antigen combining site) for a given antigen, should result in anti-antibodies (anti-idiotypes}, some of which share with the antigen a complementary structure to the paratope. Those anti-idiotypes could then possibly act as antigens, i.e., mimic the antigen. Thus, the immune system would cant' within it an internal image of the antigen, the anti-idiotype.
Anti-idiotypic antibodies have been studied as potential vaccines against pathogenic organisms (Kennedy, R. C. et al., 1986, Science 232:220;
Reagan, K. J. et al., 1983, J. Virol. 48:660; McNamara, M. K. et al., 1984, Science 226:1325; Sachs, D. L. et ai., 1982, J. Exp. Med. 155:1108) and malignant tumors (Chen, Z. J. et al., 1991, J. Immunol. 147(3):1082; Dunn, P.
L. et al., 1987, J. Immunol. 60:181-187; Herlyn, D. et al., 1987, Proc. Natl.
Aced. Sci. USA 84:8055; Chattopadhyay, P, et al., 1991, Cancer Res.
ANTIGENS
BACKGROUND OF THE INVENTION
Current immunotherapeutic approaches to inhibiting angiogenesis rely on the passive administration of large amounts of antibodies to angiogenesis targets such as the vitronectin receptor and vascular endothelial cell growth factor (VEGF). Limitations of this therapeutic strategy include the difficulty of administering large amounts of monoclonal antibodies to a patient and maintaining a constant high level of antibodies over a long period of time in a patient.
The present invention overcomes these problems by using active specific immunotherapy against angiogenesis target molecules to inhibit angiogenesis.
Such methods of immunotherapy against angiogenic molecutes include modification of immunogens to cause an immune response against the angiogenic molecules. Modification of the target antigen can be achieved by, for example, conjugation of immunogenic reagents to the antigen (see US
Patent Nos. 5,334,379); haptenization of the antigen (see U.S. Patent Nos.
4,778,752 and 5,290,551 ); the use of adjuvants bound to, or administered with, the target antigen; binding peptide fragments to the antigen; binding the target antigen to MHC class I and class II restricted antigens (see U.S. Patent No.
4,478,823); changing giycosylation patterns of the target antigens (see U.S.
Patent No. 5,484,735); presenting the target antigen on antigen presenting cells, such as dendritic cells (see U.S. Patent Nos. 5,580,563 and 5,788,963), among other methods.
Another method where immunity is induced to an angiogenesis target molecule is that of using an anti-idiotypic antibody bearing the internal image of the target antigen. Since the mimicry of the target "self' antigen by the anti-idiotype is likely to be imperfect, such anti-idiotypes will break tolerance to a self antigen, although administration of the self antigen is not able to do so. It has been previously shown that anti-idiotypes that bear the internal image of carcinoembryonic antigen (CEA) can induce anti-CEA antibodies in patients with colorectal carcinoma, while CEA itself cannot do so. Therefore, an alternative approach to using the actual "self' angiogenesis target antigen as a vaccine is to use an anti-idiotypic antibody that mimics the antigen, bears an internal image of the antigen, and elicits an immune response.
An anti-idiotypic antibody, termed either an AB2 or an anti-idiotype, is one produced in response to the presence of an antibody in an immunologically active system. Anti-idiotypes are antibodies directed against the antigen-combining region or variable region (known as the idiotype) of another antibody molecule.
The theory of idiotypic relationships and networks is based on the Jeme model (Jeme, N.K. (1974) Ann. Immunof. {Paris) 125C: 373; Jeme, N.K. et al. (1982) EMBO 1:234). Thus immunization with an antibody expressing a paratope (antigen combining site) for a given antigen, should result in anti-antibodies (anti-idiotypes}, some of which share with the antigen a complementary structure to the paratope. Those anti-idiotypes could then possibly act as antigens, i.e., mimic the antigen. Thus, the immune system would cant' within it an internal image of the antigen, the anti-idiotype.
Anti-idiotypic antibodies have been studied as potential vaccines against pathogenic organisms (Kennedy, R. C. et al., 1986, Science 232:220;
Reagan, K. J. et al., 1983, J. Virol. 48:660; McNamara, M. K. et al., 1984, Science 226:1325; Sachs, D. L. et ai., 1982, J. Exp. Med. 155:1108) and malignant tumors (Chen, Z. J. et al., 1991, J. Immunol. 147(3):1082; Dunn, P.
L. et al., 1987, J. Immunol. 60:181-187; Herlyn, D. et al., 1987, Proc. Natl.
Aced. Sci. USA 84:8055; Chattopadhyay, P, et al., 1991, Cancer Res.
51:3183). In animal studies, murine anti-idiotypic antibodies have demonstrated antigen-specific responses across species (xenogeneic model) {Chapman, P. B. and A. N. Houghton, 1991, J. Clin. Invest. 88:186) and within the same inbred species (syngeneic model) (Gaulton, G. N. et al., 1986, J. Immunol. 137:2930; Chen, Z. J. et al., 1991, J. Immunol.
147(3):1082;).
Although anti-idiotypic antibodies of angiogenic factors have been developed (W0/9608513, published March 21, 1996), including those for modulation of tumor progression (Ortega, N. et al., C. R. Acad. Sci. III (FRANCE), May 1996, 319(5):411-415), there has been no disclosure prior to this invention of the use of anti-idiotypic antibodies mimicking angiogenic factor receptors, i.e., integrins, and VEGF receptors such as basic FGF receptor, kdr, flk-1, or flt-1, to provide active immunity against angiogenesis. Therefore, this invention provides the first example of such anti-idiotypes being used to prevent or inhibit angiogenesis.
Blood vessels are formed by vasculogenesis, a process during which a primary capillary plexus is formed that is remodelled either by fusion or regression, and angiogenesis (also called neovascularization), a process in which vasculature is formed by new vessels sprouting from preexisting vessels and invading the developing organ. (Breier et al. 1996). Angiogenesis is an important process in the menstrual cycle in the endometrium, in pregnancy, and during neonatal growth. Angiogenesis is also important in wound healing and in the pathogenesis of a large variety of clinical diseases including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, and macular degeneration by neovascularization of the retina. These clinical manifestations associated with angiogenesis are refer-ed to as angiogenic diseases. {Folkman et al., Science, 235:442-447 (1987). Angiogenesis is generally absent in healthy adult or mature tissues, although it does occur in wound healing and in the corpus tuteum growth cycle. See, for example, Moses et al., Science, 248:1408-1410 (1990).
147(3):1082;).
Although anti-idiotypic antibodies of angiogenic factors have been developed (W0/9608513, published March 21, 1996), including those for modulation of tumor progression (Ortega, N. et al., C. R. Acad. Sci. III (FRANCE), May 1996, 319(5):411-415), there has been no disclosure prior to this invention of the use of anti-idiotypic antibodies mimicking angiogenic factor receptors, i.e., integrins, and VEGF receptors such as basic FGF receptor, kdr, flk-1, or flt-1, to provide active immunity against angiogenesis. Therefore, this invention provides the first example of such anti-idiotypes being used to prevent or inhibit angiogenesis.
Blood vessels are formed by vasculogenesis, a process during which a primary capillary plexus is formed that is remodelled either by fusion or regression, and angiogenesis (also called neovascularization), a process in which vasculature is formed by new vessels sprouting from preexisting vessels and invading the developing organ. (Breier et al. 1996). Angiogenesis is an important process in the menstrual cycle in the endometrium, in pregnancy, and during neonatal growth. Angiogenesis is also important in wound healing and in the pathogenesis of a large variety of clinical diseases including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, and macular degeneration by neovascularization of the retina. These clinical manifestations associated with angiogenesis are refer-ed to as angiogenic diseases. {Folkman et al., Science, 235:442-447 (1987). Angiogenesis is generally absent in healthy adult or mature tissues, although it does occur in wound healing and in the corpus tuteum growth cycle. See, for example, Moses et al., Science, 248:1408-1410 (1990).
Angiogenesis is required for tumor proliferation, since tumors need an adequate blood perfusion to obtain nutrients. Inhibiting angiogenesis by limiting vessel growth or selectively destroying proliferating endothelium would natrict tumor growth. Proposed methods of inhibiting angiogenesis include: (1 ) inhibition of release of "angiogenic molecules" such as basic-FGF (basic fibroblast growth factor), (2) neutralization of angiogenic molecules, such as by use of anti-basic-FGF antibodies, and (3) inhibition of endothelial cell response to angiogenic stimuli. Folkman et al., Cancer Biology, 3:89-96 (1992), have described several endothelial cell response inhibitors, including collagenase inhibitor, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-peniciNamine and gold thiomalate, vitamin D3 analogs, alpha-interferon, and others that might be used to inhibit angiogenesis. For additional proposed inhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-8 (1990), Moses et al., Science 248:1408-1410 (1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S.
Patent Nos. 5,092,885; 5,112,946; 5,192,744; and 5,202,352. Other new inhibitors of angiogenesis include angiostatin (O'Reilly et al., Cell 79:185-(Oct. 1994)) and endostatin.
The vascular endothelium is usually quiescent and its activation is tightly regulated during angiogenesis. Several factors have been implicated as possible regulators of angiogenesis in vivo. These include transforming growth factor (TGFb), acidic and basic fibroblast growth factor (aFGF and bFGF), platelet derived growth factor (PDGF), certain integrins, and vascular endothelial growth factor (VEGF) (Klagsbrun, M. and D'Amore. P. (1991 ) Annual Rev. Physiol. 53: 217-239).
VEGF, an endothelial cell-speck mitogen, acts as an angiogenesis inducer by specifically promoting the proliferation of endothelial cells. VEGF is a homodimeric glycoprotein consisting of two 23 kD subunits with structural similarity to PDGF. Four different monomeric isoforms of VEGF exist resulting from alternative splicing of mRNA. These include two memorane bound forms (VEGF206 and VEGF189) and two soluble forms (VEGF165 and VEGF121 ).
r In all human tissues except placenta, VEGF165 is the most abundant isoform.
VEGF is expressed in embryonic tissues (Breier et al., Development (Camb.) 114:521 (1992)x, macrophages, proliferating epidermal keratinocytes during wound healing ;Brown et al., J. Exp. Med., 176:1375 (1992)), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev. 13:18 {1992)). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumor lines including giioblastoma multiforme, hemangioblastoma, central nervous system neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al. (1992) Nature 359: 845-848; Plate, K. et al. (1993) Cancer Res. 53: 5822-5827;
Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159; Nakamura, S. et al.
(1992) AIDS Weekly, 13 (1 )). High levels of VEGF were also observed in hypoxia induced angiogenesis (Shweiki, D. et al. (1992) Nature 359: 843-845).
The biological response of VEGF is mediated through its high affinity VEGF
receptors which are selectively expressed on endothelial cells during embryogenesis (Millauer, B., et al. (1993) Cell 72: 835-846) and during tumor formation. VEGF receptors typically are class III receptor type tyrosine kinases characterized by having several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domains (Kaipainen et al., J. Exp. Med. 178:2077-2088 (1993)). The other two regions include a transmembrane region and a carboxy terminal intracellular catalytic domain interrupted by an insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Terman et al., Oncogene 6:1677-1683 (1991 ). VEGF receptors include flt-1, sequenced by Shibuya M. et al., Oncogene 5, 519-524 (1990); KDR, described in PCTIUS92/01300, filed February 20, 1992, and in Terman et al., Oncogene 6:1677-1683 (1991 ); and tlk-9, sequenced by Matthews W. et al. Proc. Natl. Acad. Sci. USA, 88:9026-9030 (1991 ). KDR is the human form of flk-1.
High levels of flk-1 are expressed by endothelial cells that infiltrate gliomas (Plate, K. et al., (1992) Nature 359: 845-848). Flk-1 levels are specifically upregulated by VEGF produced by human glioblastomas (Plate, K. et al.
(1993) Cancer Res. 53: 5822-5827). The finding of high levels of flk-1 expression in glioblastoma associated endothelial cells (GAEC) indicates that receptor activity is probably induced during tumor formation since flk-1 transcripts are barely detectable in normal brain endothelial cells. This upregulation is confined to the vascular endothelial cells in close proximity to the tumor. Blocking VEGF activity with neutralizing anti-VEGF monoclonal antibodies (mAbs) resulted in an inhibition of the growth of human tumor xenografts in nude mice (Kim, K. et al. (1993) Nature 362: 841-844), indicating a direct role for VEGF in tumor-related angiogenesis.
Integrins are a class of cellular receptors known to bind extracellular matrix proteins, and therefore mediate cell-cell and cell-extracellular matrix interactions, called cell adhesion events. The integrin receptors constitute a family of proteins with shared structural characteristics of non-covalent heterodimeric glycoprotein complexes formed of a and p subunits.
Angiogenesis in tissues has been shown to require integrin av~i3, and inhibitors of avp3 have been shown to inhibit angiogenesis (PCT Int'l. Application No.
PCTlUS95/03035, filed March 9, 1995). However, the use of anti-idiotypic antibodies mimicking any integrins, or any other angiogenesis targets, to inhibit angiogenesis has not been previously demonstrated.
Although angiogenesis related receptors are upregulated in tumor infiltrated vascular endothelial cells, the expression of these receptors is low in normal cells that are not associated with angiogenesis. Therefore, such normal cells would not be affected by inducing an immune response to such receptors to inhibit angiogenesis, and therefore to inhibit tumor growth.
An object of this invention is to provide a method of inhibiting an unwanted angiogenic condition, such as tumor angiogenesis, fieumatoid arthritis, diabetic retinopathy and psoriasis, by inducing an immune response in the subject against an angiogenic molecule. Another object of this invention is to provide immunogens that are capable of inducing an immune response in a subject against an angiogenic molecule.
SUMMARY OF THE INVENTION
The present invention provides a method of inhibiting an unwanted angiogenic condition in a mammal comprising treating the mammal with an effective amount of an immunogen that causes an immune response against a molecule that induces angiogenesis in the mammal.
The present invention also provides an immunogen that mimics a mammalian angiogenic molecule wherein the immunogen is not native to the mammal.
The invention also provides a method of inhibiting an unwanted angiogenic condition in a mammal comprising treating the mammal with an effective amount of a vector containing DNA that expresses an immunogen that causes an immune response against a molecule that induces angiogenesis in the mammal.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of inhibiting an unwanted angiogenic condition in a mammal. The method comprises treating the mammal with an effective amount of an immunogen that causes an immune response against a molecule that induces or regulates angiogenesis in the mammal. The unwanted angiogenic condition may be tumor growth, arthritis, macular degeneration, psoriasis, or any other pathological angiogenic condition. The animal is preferably a mammal, which may be a human or an animal typically used for experimentation, such as mice, rats or rabbits.
In addition to providing the methods of the invention, the present invention also provides the immunogens used in these methods. The immunogens of the invention unexpectedly induce an effective immune response when properly presented to the immune system. The immune response preferably inhibits, i.e. prevents, slows or stops, angiogenesis, and therefore inhibits or eliminates the pathological condition associated with angiogenesis, such as growth of tumors.
The immunogens of the invention may be any angiogenic molecule associated with the process of angiogenesis, such as, but not limited to vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and basic fibroblast growth factor (bFGF). Further, the immunogen may be receptors associated with the process of angiogenesis, for example, flk-1, flt-1, and KDR;
or integrins such as the vitronectin receptor av(i3; or vascular endothelial cadherins (VE-Cadherin-1 and VE-Cadherin-2); TIE-1, TIE-2lTek. However, these are examples of angiogenic molecules and receptors, and any angiogenic molecule or molecular target involved in angiogenesis may be utilized in the invention. The immunogen may be obtained from any animal or may be synthetically derived, providing it is substantially the same as that produced by the animal. The immunogen may also be introduced to any animal as naked DNA, or as a nucleic acid formulation, or as a plasmid containing DNA, which provide for the expression of a full length protein or a fragment thereof. (See U.S. Patent Nos. 5,589,466 and 5,630,796.) The immunogen may be a fragment of an antigen, epitope or antigenic determinant.
The immunogen used in the invention may be a small molecule not native to the mammal or a nucleic acid molecule not native to the mammal. Such small molecules or nucleic acids may be synthesized or isolated from an organism other than the mammal. The immunogen of the invention may also be a peptide molecule, or peptidomimetic, capable of illiciting an immune response against a molecules involved in angiogenesis. Such peptide molecules and peptidomimetics may be synthesized or isolated from an organism other than the mammal. Methods for screening small molecules, nuclic acid molecules, peptide molecules and peptidomimetics are well-known in the art. (See, for example, J. Biomolecular Screening, 1 (1 ), pg 27-31, 1996.) The immunogen used in the method of the invention may be an antigen that is native to the mammal, and is modified to improve immunogenicity. The term "native" is defined herein as meaning autologous or homologous to an animal. In other words, the native antigens of the invention are "self' proteins and therefore typically non-immunogenic in the animal from which they are derived. The antigen may be purified or substantially purified. The immunogens may also be not native, meaning foreign, to a mammal of the invention, such as small molecules or nucleic acid molecules that are not native to the mammal.
The immunogens of the invention are modified in various ways known in the art, such as by conjugating or genetically fusing the immunogen to an immunogenic reagent. Conjugation or fusion of the immunogen to an immunogenic reagent can stimulate an immune response to the immunogen.
The conjugates and fused molecules of this invention can be prepared by any of the known methods for coupling or fusing antigens to carriers or fusion molecules. The conjugates may also be prepared recombinantly as fusion proteins by methods well-known in the art. The preferred method of conjugation is covalent coupling whereby the antigen is bound directly to the immunogenic reagent. Preferred immunogenic reagents include polysaccharides (U.S. Patent No. 5,623,057), and peptidoglycans (U.S. Patent No. 5,153,173). These U.S. Patents, as well as all such patents presented in the instant specification, are herein incorporated by reference.
Another method of modifying the immunogens of the invention is for them to be bound or genetically fused with a cytokine, lymphokine, hormone or growth factor (U.S. Patent No. 5,334,379). Examples of such molecules include, but are not limited to, interferons, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 and IL-7 (U.S. Patent No. 5,334,379). As stated above, these U.S. Patents are herein incorporated by reference.
Another method of modifying the immunogens of the invention is haptenization (chemically linking) of the antigen. A hapten is a substance having the ability to, when coupled with a protein, elicit an immune response. The immunogen of the invention can itself be haptenized , or can be bound to hapten-modfied proteins. (U.S. Patent Nos. 4,778,752 and 5,290,551 ).
An additional method of modifying the immunogens of the invention is glycosylation of the antigens or glycosyiation of the carrier molecules of the antigens (see U.S. Patent Nos. 5,484,735 and 4,629,692).
Furthermore, peptidomimetic compounds, i.e., compounds which mimic the activity of peptides, may be used in modification of the immunogens of the invention (U.S. Patent Nos. 5,386,011 and 5,153,173). Additionally, peptidomimetics, which are immunogenic and illicit an immune response against molecules involved in angiogenesis are themselves useful as immunogens in the invention.
Modification of the immunogens of the invention by bonding the immunogen with a Major Histocompatibility Complex (MHC) antigen, forming a complex that is also useful in this invention. (U.S. Patent No. 4,478,823). The source of such MHC antigens and the methods of bonding the immunogens of the invention to the MHC antigens are detailed in the cited U.S. Patent No.
4,478,823, which is herein incorporated by reference.
PREPARATION OF IMMUNOGENS
Many of the angiogenic protein molecules of the invention are known, and may be obtained in natural or recombinant form by known methods. Newly identified, as well as molecules not yet identified may also be obtained and used as part of the invention. Methods of obtaining such molecules include isolating the angiogenic protein directly from cells; isolating or synthesizing DNA
encoding the protein and using the DNA to produce recombinant protein; and synthesizing the protein chemically from individual amino acids.
The entire angiogenic molecule gene or fragments of the gene may, for example, be isolated by using the known DNA sequence to construct oligonucieotide probes. To do so, DNA restriction fragments are identfied by Southern hybridization using labelled oligonucleotide probes derived from the known sequence.
Altemativeiy, DNA encoding the angiogenic molecule may be synthesized from individual nucleotides. Known methods for synthesizing DNA include preparing overlapping double-stranded oligonucleotides, filling in the gaps, and ligating the ends together.
The DNA prepared as described above may be amplified by polymerase chain reaction (PCR). Alternatively, the DNA may be amplfied by insertion into a cloning vector, which is transfected into a suitable host cell, from which the DNA may be recovered. See, generally, Sambrook et al, "Molecular Cloning,"
Second Edition, Cold Spring Harbor Laboratory Press (1987).
Recombinant methods well known in the art may be used for preparing the protein. Briefly, the angiogenic molecule-encoding DNA is inserted into an expression vector, which is transfected into a suitable host. The DNA is expressed, and the protein is harvested. See Sambrook et al., Id.
Equivalents of the angiogenic protein may also be used in the invention. Such equivalents include analogs that induce an immune response comparable to that of the protein. In addition, such equivalents are immunologically cross-reactive with their con-esponding protein. The equivalent may, for example, be a fragment of the protein, or a substitution, addition or deletion mutant of the protein.
The angiogenic protein fragment preferably contains sufficient amino acid residues to define an epitope of the antigen. The fragment may, for example, be a minigene encoding only the epitope. Methods for isolating and identifying immunogenic fragments from known immunogenic proteins are described by Salfeld et al. in J. Viral. 63, 798-808 (1989) and by Isola et al. in J.
Viral. 63, 2325-2334 (1989).
WO 99/4501$ PCTNS99/05164 If the fragment defines a suitable epitope, but is too short to be immunogenic, it _ may be conjugated to a carrier molecule. Some suitable carrier molecules include keyhole limpet hemocyanin, Ig sequences, TrpE, and human or bovine serum albumen. Conjugation may be carried out by methods known in the art.
One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.
Equivalent proteins have equivalent amino acid sequences. An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by one or more substitutions, additions and/or deletions, is considered to be an equivalent sequence. Preferably, less than 25%, more preferably less than 10%, and most preferably less than 5% of the number of amino acid residues in a sequence are substituted for, added to, or deleted from the proteins of the invention.
For example, it is known to substitute amino acids in a sequence with equivalent amino acids. Groups of amino acids generally considered to be equivalent are:
(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);
(b) Asn{N) Asp(D) Glu(E) Gln(Q);
(c) His(H) Arg{R) Lys(K);
(d) Met(M) Leu(L) Ile(I) Val(V); and (e) Phe(F) Tyr(Y) Trp(W).
The present invention also provides anti-idiotypic antibodies or fragments thereof that mimic angiogenic molecules. An "antibody" in accordance with the present specification is defined broadly as a protein that specifically binds to an epitope. The antibodies may be polyclonal, or, preferably, monoclonal. The angiogenic molecule may be any molecule involved in the process of angiogenesis, such as, but not limited to, vitronectin, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF), flt-1, flk-1, KDR, angiopoietin 1 and 2, VE-Cadherin 1 and 2, TIE-1 and TIE-?JTEK.
The modified immunogens and anti-idiotypic antibodies of the invention elicit an immune response in an animal against angiogenesis. The animal is preferably a mammal, such as a rabbit, rat, or mouse. Preferably, the animal is a human. An immune response means production of antibodies, i.e. humoral, and/or a cell-mediated response, such as a T-cell response including helper and cytotoxic T cell responses.
The anti-idiotypic antibody of the invention is directed against any antibody, which is itself directed against antigenic determinants of receptors to angiogenic molecules, or the angiogenic molecule itself. Such antibodies are known in the art, such as the anti-flk-1 antibody, DC101, described in PCT/US95/01678, filed February 10, 1995; or anti-av~i3 antibody, i.e. LM609 monoclonal antibody described in PCT Int'I. Application No. PCTIUS95103035, filed March 9, 1995. Such angiogenic molecules, such as VEGF, are also known. Further, anti-receptor and anti-angiogenic molecule antibodies may be obtained by methods known in the art such as those described below.
PREPARATION OP ANTIBODIES
The polydonal and monoclonal antibodies of the invention may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256, 495-497 ( s 975) and Campbell in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al., Eds., Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier science Publishers, Amsterdam (9985); as well as by the recombinant DNA method described by Huse et al in Science 246, 1275-1281 (1989).
The antibody may be prepared in any mammal, including mice, rats, rabbits, goats and humans. The antibody may be a member of one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
In one embodiment of the invention, a panel of syngeneic anti-idiotypic monoclonal antibodies is developed using DC101 mAb and anti-av(i3 mAb, which are both monoclonal antibodies to angiogenic receptors. An anti-idiotypic monoclonal antibody bearing the internal image of the determinants recognized by the DC101 monoclonal antibody and anti-av~i3 monoclonal antibody is identified by determining which anti-idiotypic monoclonal antibody can induce anti-avi3s mAb and anti-Flk-1 antibodies in the test animal.
Further, one embodiment of the invention provides a cell which produces the anti-idiotypic antibody of the invention. This cell may be any cell, including genetically engineered bacterial cells such as E. coli cells containing DNA to produce the antibody as well as the more typical mammalian cells such as B
cells hybridized with murine myeloma cell lines using standard fusion procedures (Keamey, J. F. et al., 1981, Eur. J. Immunol. 11:877). Methods for producing hybridomas, which have the capacity to produce a monoclonal antibody, are well known in the art. (see Niman et al. Proc. Natl. Acad. Sci.
USA 80:4949-4953 (1983) and Galfre et al., Meth. Enzymol., 73:3-46 (1981)).
Functional Eauivalents of Antibodies The invention also includes functional equivalents of the antibodies described in this specification. Functional equivalents have binding characteristics comparable to those of the antibodies, and include, for example, chimerized, humanized and single chain antibodies as well as fragments thereof.
Diabodies may also be functional equivalents of the antibodies of this invention.
Methods of producing such functional equivalents are disclosed in PCT
Application No. WO 93121319, European Patent Application No. EPO 239,400;
PCT Application Wo 89/09622; European Patent Application No. 338,745; and European Patent Application EPO 332,424.
Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. "Substantially the WO 99/45018 ,~ 5 PCT/US99/05164 same" amino acid sequence is defined herein as a sequence with at least 70%
percent homology to an amino acid sequence of an antibody of the invention, as determined by the FASTA search method in accordance with Pearson and t_ipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).
Chimerized antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human.
Humanized antibodies preferably have constant regions and variable regions other than the complementarity determining regions (CDRs) derived substantially or exclusively from the corresponding human antibody regions and CDRs derived substantially or exclusively from a mammal other than a human.
Suitable mammals other than a human include any mammal from which monoclonal antibodies may be made, such as a rabbit, rat, mouse, horse, goat, or primate.
Single chain antibodies or Fv fragments are polypeptides which consist of the V
region of the heavy chain of the antibody linked to the V region of the light chain with or without an interconnecting linker. This comprises the entire antibody combining site, and is the minimal antibody binding site. These chains may be produced in bacteria.
Functional equivalents further include fragments of antibodies that have the same or binding characteristics comparable to those of the whole antibody.
Such fragments may contain one or both Fab fragments or the F(ab')2 fragment. Preferably the antibody fragments contain all six cornplementarity determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or five CDRs, may also be functional.
Diabodies are examples of additional functional equivalents. A diabody is an antibody fragment which has two antigen binding sites and can be a bivalent or bispec~c fragment. Bispecific diabodies are heterodimers of two 'crossover' scFv fragments in which the variable light and variable heavy domains of the two antibodies are present on different polypeptide chains. (Carter and Merchant, Current Opinions in Biotechnology (8):449-454, 1997.) Further, the functional equivalents may be or may combine members of any one of the following imrnunogiobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
Gene Theraav and Vector Deliverv Intracellularly expressed antibodies, referred to as "intrabodies" can be designed to bind and inactivate target molecules inside cells. The genes encoding can be expressed intracellularly. The specific and high-affinity binding properties of antibodies, combined with their ability to be stably expressed in precise intracellular locations inside mammalian cells, provides molecules for gene therapy applications. (Marasco, W., Gene Ther (4) 1, p11-5, 1997).
Genes encoding immunogens not native to the mammal may be introduced into mammalian cells, particularly endothelial cells, by methods known in the art. Such methods have been described, for example, in Mulligan, et al., U.S.
patent 5,674,722. The methods described in Mulligan, et al., U.S. patent 5,674,722 for preparing vectors useful for introducing genes into mammalian cells, particularly endothelial cells, are incorporated herein by reference.
The immunogen may be presented to the immune system by a vehicle. For example, the immunogen may be present on the surface of an antigen presenting cell, such as a dendritic cell, or combined with a pharmaceutically acceptable carrier or adjuvant.
Antigen presenting cells are generally eukaryotic cells with major histocompatibility complex (MHC), either Class I or Class It, gene products at their cell surface. For the purposes of this spec~cation, antigen presenting cells also include recombinant eucaryotic cells, such as peripheral blood cells, and recombinant bacterial cells. Some examples of antigen presenting cells as defined by this specification include dendritic cells, macrophages that are preferably MHC Class II positive, monocytes that are preferably MHC Class II
positive, and lymphocytes. (Also see U.S. Patent No. 5,597,563).
In one embodiment of the subject invention, the antigen presenting cell is a recombinant eucaryotic cell that expresses exogenous DNA encoding the antigen of the invention. The recombinant eucaryotic cell may be prepared in vivo or in vitro.
Suitable cloning/expression vectors for inserting DNA into eucaryotic cells include well-known derivatives of SV-40, adenovirus, cytomegalovirus (CMV), and retrovirus-derived DNA sequences. Any such vectors, when coupled with vectors derived from a combination of plasmids and phage DNA, i.e. shuttle vectors, allow for the cloning and/or expression of protein coding sequences in both procaryotic and eucaryotic cells.
Other eucaryotic expression vectors are known in the art, e.g., P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S. Subramani et al, Mol.
Cell. Biol. 1, 854-864 (1981 ); R.J. Kaufmann and P.A. Sharp, "Amplification And Expression Of Sequences Cotransfected with A Modular Dihydrofoiate Reductase Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); R.J.
Kaufmann and P.A. Sharp, Mol. Cell. Biol. 159, 601-664 (1982); S.I. Scahill et al, "Expression and Characterization of the Product of a Human Immune Interferon DNA Gene in Chinese Hamster Ovary Cells," Proc. Nab. Acad. Sci.
USA 80, 4654-4659 (1983); G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci.
USA 77, 4216-4220, (1980).
The immunogens of the invention may also be presented to the immune system on the surface of recombinant bacterial cells. A suitable recombinant backerial cell is an aviruient strain of Mycobacterium bovis, such as baalle Calmette-Guerin (BCG), or an avirulent strain of Salmonella, such as S.
typhimurium. The recombinant bacterial cells may be prepared by cloning DNA
comprising the active portion of the antigen in an avirufent strain, as is known in the art; see, for example, Curtiss et al., Vaccine 6_, 155-160 (1988) and Galan et al., Gene 94, 29-35 (1990) for preparing recombinant Salmonella and Stover, C.K. et al., Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 393-398 (1991 ) for preparing recombinant BCG.
Cloning vectors may comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences. Some suitable prokaryotic cloning vectors include plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA
such as M13, fd, and other filamentous single-stranded DNA phages.
Vectors for expressing proteins in bacteria, especially E. coli, are also known.
Such vectors include the pK233 (or any of the tac family of plasmids), T7, and lambda P~. Examples of vectors that express fusion proteins are PATH vectors described by Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985). These vectors contain DNA sequences that encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy terminus. Other expression vector systems are based on beta-galactosidase (pEX); lambda P~;
maltose binding protein (pMAL); glutathione S-transferase (pGST) - see Gene 67, 31 (1988) and Peptide Research 3, 167 (1990).
The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence.
Examples of useful expression control sequences are the iac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters of SV40, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and their viruses or combinations thereof.
The immunogens of the invention may also be combined with a suitable medium. Suitable media include pharmaceutically acceptable carriers, such as phosphate buffered saline solution, liposomes and emulsions.
The immunogens may also be combined with pharmaceutically acceptable adjuvants that may enhance the immune response, such as muramyl peptides, lymphokines, such as interferon, interieukin-1 and interleukin-6, or bacterial adjuvants. The adjuvant may comprise suitable particles onto which the immunogen is adsorbed, such as aluminum oxide particles. These compositions containing adjuvants may be prepared as is known in the art.
An example of a bacterial adjuvant is BCG. When used as an antigen presenting cell as described above, recombinant BCG may additionally act as its own adjuvant. In this case, additional adjuvant may not be needed although one or more additional adjuvants may optionally be present. When used in its natural (non-recombinant) state, BCG acts solely as an adjuvant by being combined with the immunogen or anti-idiotypic antibody, resulting in a form that induces an effective immune response.
The immunogen or immunogen composition may be administered to a mammal by methods known in the art. Such methods include, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.
Assays for Determining the Level of Antibody to Anti-Receptor Antibody or Receptor in Cells The level of anti-receptor antibody or to receptor in a sample may be determined by assays known in the art using the anti-idiotypic antibody of the WO 99/45018 z~ PCT/US991051b4 invention. The results of these assays may be used for diagnostic purposes.
The target antibody or antigen may be immobilized on a support either - indirectly by using an anti-target antibody or directly to the support.
Since the anti-idiotypic antibody mimics the receptor and will compete with and thereby inhibit the ability of the receptor to bind with anti-receptor antibodies, a competitive assay may be used to measure the concentration of receptor or anti-receptor antibodies by correlating the level of anti-idiotypic antibody binding to the concentration of receptor or anti-receptor antibodies.
A variety of assays are available for detecting proteins with labeled antibodies. In a one-step assay, the target molecule, if it is present, is immobilized and incubated with a labeled anti-idiotypic antibody. The labeled anti-idiotypic antibody binds to the immobilized target molecule. After washing to remove unbound molecules, the sample is assayed for the presence of the label.
In a two-step assay, immobilized target molecule is incubated with an unlabeled anti-idiotypic antibody. The target molecule-unlabeled anti-idiotypic antibody complex, if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody. The sample is washed and assayed for the presence of the label, as described above.
The choice of marker used to label the antibodies will vary depending upon the application. However, the choice of marker is readily determinable to one skilled in the art. The labeled antibodies may be polycional or monoclonal. In a preferred embodiment, the antibody is monoclonal.
Purification Methods The invention also provides a method of purifying anti-receptor antibodies in a sample comprising contacting the sample with an anti-idiotypic antibody of the invention, isolating the complex between anti-receptor antibodies from the sample and the anti-idiotypic antibody, and recovering the anti-receptor antibodies from the complex. The anti-idiotypic antibody is preferably immobilized.
Experimental Examples Dendritic cells (DC): DC were isolated using the following protocol. Bone marrow from C57BL6 mice was depleted of red blood cells by treatment in 0.5% amonium chloride, followed by treatment with a combination of monoclonal antibodies specific for CD4, CD8, la, granulocytes and then with rabbit complement. The remaining cells were cultured in RPMI 1640 containing 10% FBS as well as GM-CSF and IL-4. Three days later, nonadherent cells were discarded and adherent cells cultured for 4 more days. Adherent cells were then transferred to a new plate and cultured for another 3 days before harvesting for use as antigen presenting cells.
Pulsing of DC with Flk-1AP antigen: DC were washed twice in serum-free AIMV media and incubated overnight in AIMV with 100uglml of an affinity-purified fusion protein of Flk-1 and alkaline phosphatase (Flk-1AP). The cells were then washed twice in AIMV before being used for vaccination.
Vaccination of mice with DC pulsed with Flk-1AP: Lewis Lung carcinoma metastasis model was used to assess the antitumor effect of vaccination with DC pulsed with Flk-1AP. Briefly, C57BL6 mice were injected either intravenously or intraperiteneally, with each mouse receiving 1 x 105 Flk-1 AP
pulsed DC at Day 0. Seven days later, each mouse was inoculated intra-footpad with 1x 105 D122 cells. Visible tumors were surgically removed at Day 10. During this period, mice received two boost vaccinations every ten days. At Day 60, mice were sacrificed and lungs removed for weighing and assessment of metastases. As a control, a group of mice were vaccinated with DC alone.
Experimental Results Inhibition of tumor metastases: Mice vaccinated with DC pulsed with Flk-1AP developed a significantly smaller number of tumors in the lungs compared to the groups vaccinated with DC alone. Comparably, the average - weight of the lungs from the Flk-AP group was significantly lower than the controls. In both instances, the number of tumors present and the weight of the lungs of mice vaccinated with DC pulsed with Flk-1AP was less than half of the control group.
Patent Nos. 5,092,885; 5,112,946; 5,192,744; and 5,202,352. Other new inhibitors of angiogenesis include angiostatin (O'Reilly et al., Cell 79:185-(Oct. 1994)) and endostatin.
The vascular endothelium is usually quiescent and its activation is tightly regulated during angiogenesis. Several factors have been implicated as possible regulators of angiogenesis in vivo. These include transforming growth factor (TGFb), acidic and basic fibroblast growth factor (aFGF and bFGF), platelet derived growth factor (PDGF), certain integrins, and vascular endothelial growth factor (VEGF) (Klagsbrun, M. and D'Amore. P. (1991 ) Annual Rev. Physiol. 53: 217-239).
VEGF, an endothelial cell-speck mitogen, acts as an angiogenesis inducer by specifically promoting the proliferation of endothelial cells. VEGF is a homodimeric glycoprotein consisting of two 23 kD subunits with structural similarity to PDGF. Four different monomeric isoforms of VEGF exist resulting from alternative splicing of mRNA. These include two memorane bound forms (VEGF206 and VEGF189) and two soluble forms (VEGF165 and VEGF121 ).
r In all human tissues except placenta, VEGF165 is the most abundant isoform.
VEGF is expressed in embryonic tissues (Breier et al., Development (Camb.) 114:521 (1992)x, macrophages, proliferating epidermal keratinocytes during wound healing ;Brown et al., J. Exp. Med., 176:1375 (1992)), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev. 13:18 {1992)). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumor lines including giioblastoma multiforme, hemangioblastoma, central nervous system neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al. (1992) Nature 359: 845-848; Plate, K. et al. (1993) Cancer Res. 53: 5822-5827;
Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159; Nakamura, S. et al.
(1992) AIDS Weekly, 13 (1 )). High levels of VEGF were also observed in hypoxia induced angiogenesis (Shweiki, D. et al. (1992) Nature 359: 843-845).
The biological response of VEGF is mediated through its high affinity VEGF
receptors which are selectively expressed on endothelial cells during embryogenesis (Millauer, B., et al. (1993) Cell 72: 835-846) and during tumor formation. VEGF receptors typically are class III receptor type tyrosine kinases characterized by having several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domains (Kaipainen et al., J. Exp. Med. 178:2077-2088 (1993)). The other two regions include a transmembrane region and a carboxy terminal intracellular catalytic domain interrupted by an insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Terman et al., Oncogene 6:1677-1683 (1991 ). VEGF receptors include flt-1, sequenced by Shibuya M. et al., Oncogene 5, 519-524 (1990); KDR, described in PCTIUS92/01300, filed February 20, 1992, and in Terman et al., Oncogene 6:1677-1683 (1991 ); and tlk-9, sequenced by Matthews W. et al. Proc. Natl. Acad. Sci. USA, 88:9026-9030 (1991 ). KDR is the human form of flk-1.
High levels of flk-1 are expressed by endothelial cells that infiltrate gliomas (Plate, K. et al., (1992) Nature 359: 845-848). Flk-1 levels are specifically upregulated by VEGF produced by human glioblastomas (Plate, K. et al.
(1993) Cancer Res. 53: 5822-5827). The finding of high levels of flk-1 expression in glioblastoma associated endothelial cells (GAEC) indicates that receptor activity is probably induced during tumor formation since flk-1 transcripts are barely detectable in normal brain endothelial cells. This upregulation is confined to the vascular endothelial cells in close proximity to the tumor. Blocking VEGF activity with neutralizing anti-VEGF monoclonal antibodies (mAbs) resulted in an inhibition of the growth of human tumor xenografts in nude mice (Kim, K. et al. (1993) Nature 362: 841-844), indicating a direct role for VEGF in tumor-related angiogenesis.
Integrins are a class of cellular receptors known to bind extracellular matrix proteins, and therefore mediate cell-cell and cell-extracellular matrix interactions, called cell adhesion events. The integrin receptors constitute a family of proteins with shared structural characteristics of non-covalent heterodimeric glycoprotein complexes formed of a and p subunits.
Angiogenesis in tissues has been shown to require integrin av~i3, and inhibitors of avp3 have been shown to inhibit angiogenesis (PCT Int'l. Application No.
PCTlUS95/03035, filed March 9, 1995). However, the use of anti-idiotypic antibodies mimicking any integrins, or any other angiogenesis targets, to inhibit angiogenesis has not been previously demonstrated.
Although angiogenesis related receptors are upregulated in tumor infiltrated vascular endothelial cells, the expression of these receptors is low in normal cells that are not associated with angiogenesis. Therefore, such normal cells would not be affected by inducing an immune response to such receptors to inhibit angiogenesis, and therefore to inhibit tumor growth.
An object of this invention is to provide a method of inhibiting an unwanted angiogenic condition, such as tumor angiogenesis, fieumatoid arthritis, diabetic retinopathy and psoriasis, by inducing an immune response in the subject against an angiogenic molecule. Another object of this invention is to provide immunogens that are capable of inducing an immune response in a subject against an angiogenic molecule.
SUMMARY OF THE INVENTION
The present invention provides a method of inhibiting an unwanted angiogenic condition in a mammal comprising treating the mammal with an effective amount of an immunogen that causes an immune response against a molecule that induces angiogenesis in the mammal.
The present invention also provides an immunogen that mimics a mammalian angiogenic molecule wherein the immunogen is not native to the mammal.
The invention also provides a method of inhibiting an unwanted angiogenic condition in a mammal comprising treating the mammal with an effective amount of a vector containing DNA that expresses an immunogen that causes an immune response against a molecule that induces angiogenesis in the mammal.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of inhibiting an unwanted angiogenic condition in a mammal. The method comprises treating the mammal with an effective amount of an immunogen that causes an immune response against a molecule that induces or regulates angiogenesis in the mammal. The unwanted angiogenic condition may be tumor growth, arthritis, macular degeneration, psoriasis, or any other pathological angiogenic condition. The animal is preferably a mammal, which may be a human or an animal typically used for experimentation, such as mice, rats or rabbits.
In addition to providing the methods of the invention, the present invention also provides the immunogens used in these methods. The immunogens of the invention unexpectedly induce an effective immune response when properly presented to the immune system. The immune response preferably inhibits, i.e. prevents, slows or stops, angiogenesis, and therefore inhibits or eliminates the pathological condition associated with angiogenesis, such as growth of tumors.
The immunogens of the invention may be any angiogenic molecule associated with the process of angiogenesis, such as, but not limited to vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and basic fibroblast growth factor (bFGF). Further, the immunogen may be receptors associated with the process of angiogenesis, for example, flk-1, flt-1, and KDR;
or integrins such as the vitronectin receptor av(i3; or vascular endothelial cadherins (VE-Cadherin-1 and VE-Cadherin-2); TIE-1, TIE-2lTek. However, these are examples of angiogenic molecules and receptors, and any angiogenic molecule or molecular target involved in angiogenesis may be utilized in the invention. The immunogen may be obtained from any animal or may be synthetically derived, providing it is substantially the same as that produced by the animal. The immunogen may also be introduced to any animal as naked DNA, or as a nucleic acid formulation, or as a plasmid containing DNA, which provide for the expression of a full length protein or a fragment thereof. (See U.S. Patent Nos. 5,589,466 and 5,630,796.) The immunogen may be a fragment of an antigen, epitope or antigenic determinant.
The immunogen used in the invention may be a small molecule not native to the mammal or a nucleic acid molecule not native to the mammal. Such small molecules or nucleic acids may be synthesized or isolated from an organism other than the mammal. The immunogen of the invention may also be a peptide molecule, or peptidomimetic, capable of illiciting an immune response against a molecules involved in angiogenesis. Such peptide molecules and peptidomimetics may be synthesized or isolated from an organism other than the mammal. Methods for screening small molecules, nuclic acid molecules, peptide molecules and peptidomimetics are well-known in the art. (See, for example, J. Biomolecular Screening, 1 (1 ), pg 27-31, 1996.) The immunogen used in the method of the invention may be an antigen that is native to the mammal, and is modified to improve immunogenicity. The term "native" is defined herein as meaning autologous or homologous to an animal. In other words, the native antigens of the invention are "self' proteins and therefore typically non-immunogenic in the animal from which they are derived. The antigen may be purified or substantially purified. The immunogens may also be not native, meaning foreign, to a mammal of the invention, such as small molecules or nucleic acid molecules that are not native to the mammal.
The immunogens of the invention are modified in various ways known in the art, such as by conjugating or genetically fusing the immunogen to an immunogenic reagent. Conjugation or fusion of the immunogen to an immunogenic reagent can stimulate an immune response to the immunogen.
The conjugates and fused molecules of this invention can be prepared by any of the known methods for coupling or fusing antigens to carriers or fusion molecules. The conjugates may also be prepared recombinantly as fusion proteins by methods well-known in the art. The preferred method of conjugation is covalent coupling whereby the antigen is bound directly to the immunogenic reagent. Preferred immunogenic reagents include polysaccharides (U.S. Patent No. 5,623,057), and peptidoglycans (U.S. Patent No. 5,153,173). These U.S. Patents, as well as all such patents presented in the instant specification, are herein incorporated by reference.
Another method of modifying the immunogens of the invention is for them to be bound or genetically fused with a cytokine, lymphokine, hormone or growth factor (U.S. Patent No. 5,334,379). Examples of such molecules include, but are not limited to, interferons, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 and IL-7 (U.S. Patent No. 5,334,379). As stated above, these U.S. Patents are herein incorporated by reference.
Another method of modifying the immunogens of the invention is haptenization (chemically linking) of the antigen. A hapten is a substance having the ability to, when coupled with a protein, elicit an immune response. The immunogen of the invention can itself be haptenized , or can be bound to hapten-modfied proteins. (U.S. Patent Nos. 4,778,752 and 5,290,551 ).
An additional method of modifying the immunogens of the invention is glycosylation of the antigens or glycosyiation of the carrier molecules of the antigens (see U.S. Patent Nos. 5,484,735 and 4,629,692).
Furthermore, peptidomimetic compounds, i.e., compounds which mimic the activity of peptides, may be used in modification of the immunogens of the invention (U.S. Patent Nos. 5,386,011 and 5,153,173). Additionally, peptidomimetics, which are immunogenic and illicit an immune response against molecules involved in angiogenesis are themselves useful as immunogens in the invention.
Modification of the immunogens of the invention by bonding the immunogen with a Major Histocompatibility Complex (MHC) antigen, forming a complex that is also useful in this invention. (U.S. Patent No. 4,478,823). The source of such MHC antigens and the methods of bonding the immunogens of the invention to the MHC antigens are detailed in the cited U.S. Patent No.
4,478,823, which is herein incorporated by reference.
PREPARATION OF IMMUNOGENS
Many of the angiogenic protein molecules of the invention are known, and may be obtained in natural or recombinant form by known methods. Newly identified, as well as molecules not yet identified may also be obtained and used as part of the invention. Methods of obtaining such molecules include isolating the angiogenic protein directly from cells; isolating or synthesizing DNA
encoding the protein and using the DNA to produce recombinant protein; and synthesizing the protein chemically from individual amino acids.
The entire angiogenic molecule gene or fragments of the gene may, for example, be isolated by using the known DNA sequence to construct oligonucieotide probes. To do so, DNA restriction fragments are identfied by Southern hybridization using labelled oligonucleotide probes derived from the known sequence.
Altemativeiy, DNA encoding the angiogenic molecule may be synthesized from individual nucleotides. Known methods for synthesizing DNA include preparing overlapping double-stranded oligonucleotides, filling in the gaps, and ligating the ends together.
The DNA prepared as described above may be amplified by polymerase chain reaction (PCR). Alternatively, the DNA may be amplfied by insertion into a cloning vector, which is transfected into a suitable host cell, from which the DNA may be recovered. See, generally, Sambrook et al, "Molecular Cloning,"
Second Edition, Cold Spring Harbor Laboratory Press (1987).
Recombinant methods well known in the art may be used for preparing the protein. Briefly, the angiogenic molecule-encoding DNA is inserted into an expression vector, which is transfected into a suitable host. The DNA is expressed, and the protein is harvested. See Sambrook et al., Id.
Equivalents of the angiogenic protein may also be used in the invention. Such equivalents include analogs that induce an immune response comparable to that of the protein. In addition, such equivalents are immunologically cross-reactive with their con-esponding protein. The equivalent may, for example, be a fragment of the protein, or a substitution, addition or deletion mutant of the protein.
The angiogenic protein fragment preferably contains sufficient amino acid residues to define an epitope of the antigen. The fragment may, for example, be a minigene encoding only the epitope. Methods for isolating and identifying immunogenic fragments from known immunogenic proteins are described by Salfeld et al. in J. Viral. 63, 798-808 (1989) and by Isola et al. in J.
Viral. 63, 2325-2334 (1989).
WO 99/4501$ PCTNS99/05164 If the fragment defines a suitable epitope, but is too short to be immunogenic, it _ may be conjugated to a carrier molecule. Some suitable carrier molecules include keyhole limpet hemocyanin, Ig sequences, TrpE, and human or bovine serum albumen. Conjugation may be carried out by methods known in the art.
One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.
Equivalent proteins have equivalent amino acid sequences. An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by one or more substitutions, additions and/or deletions, is considered to be an equivalent sequence. Preferably, less than 25%, more preferably less than 10%, and most preferably less than 5% of the number of amino acid residues in a sequence are substituted for, added to, or deleted from the proteins of the invention.
For example, it is known to substitute amino acids in a sequence with equivalent amino acids. Groups of amino acids generally considered to be equivalent are:
(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);
(b) Asn{N) Asp(D) Glu(E) Gln(Q);
(c) His(H) Arg{R) Lys(K);
(d) Met(M) Leu(L) Ile(I) Val(V); and (e) Phe(F) Tyr(Y) Trp(W).
The present invention also provides anti-idiotypic antibodies or fragments thereof that mimic angiogenic molecules. An "antibody" in accordance with the present specification is defined broadly as a protein that specifically binds to an epitope. The antibodies may be polyclonal, or, preferably, monoclonal. The angiogenic molecule may be any molecule involved in the process of angiogenesis, such as, but not limited to, vitronectin, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF), flt-1, flk-1, KDR, angiopoietin 1 and 2, VE-Cadherin 1 and 2, TIE-1 and TIE-?JTEK.
The modified immunogens and anti-idiotypic antibodies of the invention elicit an immune response in an animal against angiogenesis. The animal is preferably a mammal, such as a rabbit, rat, or mouse. Preferably, the animal is a human. An immune response means production of antibodies, i.e. humoral, and/or a cell-mediated response, such as a T-cell response including helper and cytotoxic T cell responses.
The anti-idiotypic antibody of the invention is directed against any antibody, which is itself directed against antigenic determinants of receptors to angiogenic molecules, or the angiogenic molecule itself. Such antibodies are known in the art, such as the anti-flk-1 antibody, DC101, described in PCT/US95/01678, filed February 10, 1995; or anti-av~i3 antibody, i.e. LM609 monoclonal antibody described in PCT Int'I. Application No. PCTIUS95103035, filed March 9, 1995. Such angiogenic molecules, such as VEGF, are also known. Further, anti-receptor and anti-angiogenic molecule antibodies may be obtained by methods known in the art such as those described below.
PREPARATION OP ANTIBODIES
The polydonal and monoclonal antibodies of the invention may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256, 495-497 ( s 975) and Campbell in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al., Eds., Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier science Publishers, Amsterdam (9985); as well as by the recombinant DNA method described by Huse et al in Science 246, 1275-1281 (1989).
The antibody may be prepared in any mammal, including mice, rats, rabbits, goats and humans. The antibody may be a member of one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
In one embodiment of the invention, a panel of syngeneic anti-idiotypic monoclonal antibodies is developed using DC101 mAb and anti-av(i3 mAb, which are both monoclonal antibodies to angiogenic receptors. An anti-idiotypic monoclonal antibody bearing the internal image of the determinants recognized by the DC101 monoclonal antibody and anti-av~i3 monoclonal antibody is identified by determining which anti-idiotypic monoclonal antibody can induce anti-avi3s mAb and anti-Flk-1 antibodies in the test animal.
Further, one embodiment of the invention provides a cell which produces the anti-idiotypic antibody of the invention. This cell may be any cell, including genetically engineered bacterial cells such as E. coli cells containing DNA to produce the antibody as well as the more typical mammalian cells such as B
cells hybridized with murine myeloma cell lines using standard fusion procedures (Keamey, J. F. et al., 1981, Eur. J. Immunol. 11:877). Methods for producing hybridomas, which have the capacity to produce a monoclonal antibody, are well known in the art. (see Niman et al. Proc. Natl. Acad. Sci.
USA 80:4949-4953 (1983) and Galfre et al., Meth. Enzymol., 73:3-46 (1981)).
Functional Eauivalents of Antibodies The invention also includes functional equivalents of the antibodies described in this specification. Functional equivalents have binding characteristics comparable to those of the antibodies, and include, for example, chimerized, humanized and single chain antibodies as well as fragments thereof.
Diabodies may also be functional equivalents of the antibodies of this invention.
Methods of producing such functional equivalents are disclosed in PCT
Application No. WO 93121319, European Patent Application No. EPO 239,400;
PCT Application Wo 89/09622; European Patent Application No. 338,745; and European Patent Application EPO 332,424.
Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. "Substantially the WO 99/45018 ,~ 5 PCT/US99/05164 same" amino acid sequence is defined herein as a sequence with at least 70%
percent homology to an amino acid sequence of an antibody of the invention, as determined by the FASTA search method in accordance with Pearson and t_ipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).
Chimerized antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human.
Humanized antibodies preferably have constant regions and variable regions other than the complementarity determining regions (CDRs) derived substantially or exclusively from the corresponding human antibody regions and CDRs derived substantially or exclusively from a mammal other than a human.
Suitable mammals other than a human include any mammal from which monoclonal antibodies may be made, such as a rabbit, rat, mouse, horse, goat, or primate.
Single chain antibodies or Fv fragments are polypeptides which consist of the V
region of the heavy chain of the antibody linked to the V region of the light chain with or without an interconnecting linker. This comprises the entire antibody combining site, and is the minimal antibody binding site. These chains may be produced in bacteria.
Functional equivalents further include fragments of antibodies that have the same or binding characteristics comparable to those of the whole antibody.
Such fragments may contain one or both Fab fragments or the F(ab')2 fragment. Preferably the antibody fragments contain all six cornplementarity determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or five CDRs, may also be functional.
Diabodies are examples of additional functional equivalents. A diabody is an antibody fragment which has two antigen binding sites and can be a bivalent or bispec~c fragment. Bispecific diabodies are heterodimers of two 'crossover' scFv fragments in which the variable light and variable heavy domains of the two antibodies are present on different polypeptide chains. (Carter and Merchant, Current Opinions in Biotechnology (8):449-454, 1997.) Further, the functional equivalents may be or may combine members of any one of the following imrnunogiobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
Gene Theraav and Vector Deliverv Intracellularly expressed antibodies, referred to as "intrabodies" can be designed to bind and inactivate target molecules inside cells. The genes encoding can be expressed intracellularly. The specific and high-affinity binding properties of antibodies, combined with their ability to be stably expressed in precise intracellular locations inside mammalian cells, provides molecules for gene therapy applications. (Marasco, W., Gene Ther (4) 1, p11-5, 1997).
Genes encoding immunogens not native to the mammal may be introduced into mammalian cells, particularly endothelial cells, by methods known in the art. Such methods have been described, for example, in Mulligan, et al., U.S.
patent 5,674,722. The methods described in Mulligan, et al., U.S. patent 5,674,722 for preparing vectors useful for introducing genes into mammalian cells, particularly endothelial cells, are incorporated herein by reference.
The immunogen may be presented to the immune system by a vehicle. For example, the immunogen may be present on the surface of an antigen presenting cell, such as a dendritic cell, or combined with a pharmaceutically acceptable carrier or adjuvant.
Antigen presenting cells are generally eukaryotic cells with major histocompatibility complex (MHC), either Class I or Class It, gene products at their cell surface. For the purposes of this spec~cation, antigen presenting cells also include recombinant eucaryotic cells, such as peripheral blood cells, and recombinant bacterial cells. Some examples of antigen presenting cells as defined by this specification include dendritic cells, macrophages that are preferably MHC Class II positive, monocytes that are preferably MHC Class II
positive, and lymphocytes. (Also see U.S. Patent No. 5,597,563).
In one embodiment of the subject invention, the antigen presenting cell is a recombinant eucaryotic cell that expresses exogenous DNA encoding the antigen of the invention. The recombinant eucaryotic cell may be prepared in vivo or in vitro.
Suitable cloning/expression vectors for inserting DNA into eucaryotic cells include well-known derivatives of SV-40, adenovirus, cytomegalovirus (CMV), and retrovirus-derived DNA sequences. Any such vectors, when coupled with vectors derived from a combination of plasmids and phage DNA, i.e. shuttle vectors, allow for the cloning and/or expression of protein coding sequences in both procaryotic and eucaryotic cells.
Other eucaryotic expression vectors are known in the art, e.g., P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S. Subramani et al, Mol.
Cell. Biol. 1, 854-864 (1981 ); R.J. Kaufmann and P.A. Sharp, "Amplification And Expression Of Sequences Cotransfected with A Modular Dihydrofoiate Reductase Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); R.J.
Kaufmann and P.A. Sharp, Mol. Cell. Biol. 159, 601-664 (1982); S.I. Scahill et al, "Expression and Characterization of the Product of a Human Immune Interferon DNA Gene in Chinese Hamster Ovary Cells," Proc. Nab. Acad. Sci.
USA 80, 4654-4659 (1983); G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci.
USA 77, 4216-4220, (1980).
The immunogens of the invention may also be presented to the immune system on the surface of recombinant bacterial cells. A suitable recombinant backerial cell is an aviruient strain of Mycobacterium bovis, such as baalle Calmette-Guerin (BCG), or an avirulent strain of Salmonella, such as S.
typhimurium. The recombinant bacterial cells may be prepared by cloning DNA
comprising the active portion of the antigen in an avirufent strain, as is known in the art; see, for example, Curtiss et al., Vaccine 6_, 155-160 (1988) and Galan et al., Gene 94, 29-35 (1990) for preparing recombinant Salmonella and Stover, C.K. et al., Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 393-398 (1991 ) for preparing recombinant BCG.
Cloning vectors may comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences. Some suitable prokaryotic cloning vectors include plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA
such as M13, fd, and other filamentous single-stranded DNA phages.
Vectors for expressing proteins in bacteria, especially E. coli, are also known.
Such vectors include the pK233 (or any of the tac family of plasmids), T7, and lambda P~. Examples of vectors that express fusion proteins are PATH vectors described by Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985). These vectors contain DNA sequences that encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy terminus. Other expression vector systems are based on beta-galactosidase (pEX); lambda P~;
maltose binding protein (pMAL); glutathione S-transferase (pGST) - see Gene 67, 31 (1988) and Peptide Research 3, 167 (1990).
The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence.
Examples of useful expression control sequences are the iac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters of SV40, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and their viruses or combinations thereof.
The immunogens of the invention may also be combined with a suitable medium. Suitable media include pharmaceutically acceptable carriers, such as phosphate buffered saline solution, liposomes and emulsions.
The immunogens may also be combined with pharmaceutically acceptable adjuvants that may enhance the immune response, such as muramyl peptides, lymphokines, such as interferon, interieukin-1 and interleukin-6, or bacterial adjuvants. The adjuvant may comprise suitable particles onto which the immunogen is adsorbed, such as aluminum oxide particles. These compositions containing adjuvants may be prepared as is known in the art.
An example of a bacterial adjuvant is BCG. When used as an antigen presenting cell as described above, recombinant BCG may additionally act as its own adjuvant. In this case, additional adjuvant may not be needed although one or more additional adjuvants may optionally be present. When used in its natural (non-recombinant) state, BCG acts solely as an adjuvant by being combined with the immunogen or anti-idiotypic antibody, resulting in a form that induces an effective immune response.
The immunogen or immunogen composition may be administered to a mammal by methods known in the art. Such methods include, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.
Assays for Determining the Level of Antibody to Anti-Receptor Antibody or Receptor in Cells The level of anti-receptor antibody or to receptor in a sample may be determined by assays known in the art using the anti-idiotypic antibody of the WO 99/45018 z~ PCT/US991051b4 invention. The results of these assays may be used for diagnostic purposes.
The target antibody or antigen may be immobilized on a support either - indirectly by using an anti-target antibody or directly to the support.
Since the anti-idiotypic antibody mimics the receptor and will compete with and thereby inhibit the ability of the receptor to bind with anti-receptor antibodies, a competitive assay may be used to measure the concentration of receptor or anti-receptor antibodies by correlating the level of anti-idiotypic antibody binding to the concentration of receptor or anti-receptor antibodies.
A variety of assays are available for detecting proteins with labeled antibodies. In a one-step assay, the target molecule, if it is present, is immobilized and incubated with a labeled anti-idiotypic antibody. The labeled anti-idiotypic antibody binds to the immobilized target molecule. After washing to remove unbound molecules, the sample is assayed for the presence of the label.
In a two-step assay, immobilized target molecule is incubated with an unlabeled anti-idiotypic antibody. The target molecule-unlabeled anti-idiotypic antibody complex, if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody. The sample is washed and assayed for the presence of the label, as described above.
The choice of marker used to label the antibodies will vary depending upon the application. However, the choice of marker is readily determinable to one skilled in the art. The labeled antibodies may be polycional or monoclonal. In a preferred embodiment, the antibody is monoclonal.
Purification Methods The invention also provides a method of purifying anti-receptor antibodies in a sample comprising contacting the sample with an anti-idiotypic antibody of the invention, isolating the complex between anti-receptor antibodies from the sample and the anti-idiotypic antibody, and recovering the anti-receptor antibodies from the complex. The anti-idiotypic antibody is preferably immobilized.
Experimental Examples Dendritic cells (DC): DC were isolated using the following protocol. Bone marrow from C57BL6 mice was depleted of red blood cells by treatment in 0.5% amonium chloride, followed by treatment with a combination of monoclonal antibodies specific for CD4, CD8, la, granulocytes and then with rabbit complement. The remaining cells were cultured in RPMI 1640 containing 10% FBS as well as GM-CSF and IL-4. Three days later, nonadherent cells were discarded and adherent cells cultured for 4 more days. Adherent cells were then transferred to a new plate and cultured for another 3 days before harvesting for use as antigen presenting cells.
Pulsing of DC with Flk-1AP antigen: DC were washed twice in serum-free AIMV media and incubated overnight in AIMV with 100uglml of an affinity-purified fusion protein of Flk-1 and alkaline phosphatase (Flk-1AP). The cells were then washed twice in AIMV before being used for vaccination.
Vaccination of mice with DC pulsed with Flk-1AP: Lewis Lung carcinoma metastasis model was used to assess the antitumor effect of vaccination with DC pulsed with Flk-1AP. Briefly, C57BL6 mice were injected either intravenously or intraperiteneally, with each mouse receiving 1 x 105 Flk-1 AP
pulsed DC at Day 0. Seven days later, each mouse was inoculated intra-footpad with 1x 105 D122 cells. Visible tumors were surgically removed at Day 10. During this period, mice received two boost vaccinations every ten days. At Day 60, mice were sacrificed and lungs removed for weighing and assessment of metastases. As a control, a group of mice were vaccinated with DC alone.
Experimental Results Inhibition of tumor metastases: Mice vaccinated with DC pulsed with Flk-1AP developed a significantly smaller number of tumors in the lungs compared to the groups vaccinated with DC alone. Comparably, the average - weight of the lungs from the Flk-AP group was significantly lower than the controls. In both instances, the number of tumors present and the weight of the lungs of mice vaccinated with DC pulsed with Flk-1AP was less than half of the control group.
Claims (56)
1. A method of inhibiting an unwanted angiogenic condition in a mammal in need thereof comprising treating the mammal with an effective amount of an immunogen that causes an immune response against a molecule that induces angiogenesis in the mammal.
2. The method of claim 1 wherein the unwanted angiogenic condition is tumor growth.
3. The method of claim 1 wherein the unwanted angiogenic condition is arthritis.
4. The method of claim 1 wherein the unwanted angiogenic condition is macular degeneration.
5. The method of claim 1 wherein the unwanted angiogenic condition is psoriasis.
6. The method of claim 1 wherein the mammal is a human.
7. The method of claim 1 wherein the immunogen is an antigen that is native to the mammal, and that is modified to improve immunogenicity.
8. The method of claim 7 wherein the antigen is a haptenized antigen.
9. The method of claim 7 wherein the antigen is conjugated to an immunogenic compound.
10. The method of claim 7 wherein the antigen is combined with an adjuvant.
11. The method of claim 1 wherein the immunogen is bound to a MHC
Class I Restricted Antigen forming a complex not native to the mammal.
Class I Restricted Antigen forming a complex not native to the mammal.
12. The method of claim 1 wherein the immunogen is bound to a MHC
Class II
Restricted Antigen forming a complex not native to the mammal.
Class II
Restricted Antigen forming a complex not native to the mammal.
13. The method of claim 7 wherein the antigen is substantially purified.
14. The method of claim 1 wherein the immunogen is an anti-idiotypic monoclonal antibody.
15. The method of claim 1 wherein the immunogen is a synthetic peptide not native to the mammal.
16. The method of claim 1 wherein the immunogen is a small molecule not native to the mammal.
17. The method of claim 1 wherein the immunogen is expressed on an antigen-presenting cell not native to the mammal.
18. The method of claim 17 wherein the antigen-presenting cell is a dendritic cell.
19. The method of claim 1 wherein the immunogen is a nucleic acid molecule not native to the mammal.
20. The method of claim 1 wherein the a molecule that induces angiogenesis is FLK-1.
21. The method of claim 1 wherein the a molecule that induces angiogenesis is KDR.
22. The method of claim 1 wherein the a molecule that induces angiogenesis is FLT-1.
23. The method of claim 1 wherein the a molecule that induces angiogenesis is VEGF.
24. The method of claim 1 wherein the a molecule that induces angiogenesis is a Vascular Endothelial Cadherin.
25. The method of claim 1 wherein the a molecule that induces angiogenesis is TIE-1.
26. The method of claim 1 wherein the a molecule that induces angiogenesis is T1E-2/Tek.
27. The method of claim 1 wherein the a molecule that induces angiogenesis is an integrin.
28. The method of claim 27 wherein the integrin is alphaVbeta3.
29. The method of claim 1 wherein the a molecule that induces angiogenesis is bFGF.
30. The method of claim 1 wherein the a molecule that induces angiogenesis is vitronectin.
31. An immunogen that mimics a mammalian angiogenic molecule wherein the immunogen not native to the mammal.
32. The immunogen of claim 31, wherein the immunogen is an anti-idiotypic antibody.
33. The immunogen of claim 31, wherein the immunogen is an isolated peptide.
34. The immunogen of claim 31, wherein the immunogen is an isolated small molecule.
35. The immunogen of claim 31, wherein the immunogen is a nucleic acid molecule.
36. The immunogen of claim 31, wherein the immunogen is an antigen-presenting cell.
37. The immunogen of claim 36, wherein the antigen-presenting cell is a dendritic cell.
38. The immunogen of claim 31, wherein the immunogen is a MHC Class I
Restricted Antigen complex.
Restricted Antigen complex.
39. The immunogen of claim 31, wherein the immunogen is a MHC Class II
Restricted Antigen complex.
Restricted Antigen complex.
40. The immunogen of claim 32, wherein the antibody is monoclonal.
41. The immunogen of claim 40, comprising a fragment of the monoclonal antibody.
42. A cell which produces the anti-idiotypic antibody of claim 40.
43. A cell of claim 42 that is a hybridoma.
44. A polypeptide comprising an amino acid sequence that is substantially the same as, the amino acid sequence of the variable region of the monoclonal antibody of claim 40, and that has the same binding specificity.
45. A nucleic acid that encodes the hypervariable region of the antibody of claim 40.
46. A nucleic acid that encodes the polypeptide of claim 44.
47. A nucleic acid that hybridizes under stringent conditions to the nucleic acid of claim 45 and that encodes a polypeptide having the same binding specificity as the hypervariable region encoded by the nucleic acid of claim 45.
48. A chimeric antibody comprising the polypeptide of claim 44.
49. A chimeric antibody of claim 48 comprising an amino acid sequence of a human antibody constant region and an amino acid sequence of a non-human antibody variable region.
50. A chimeric antibody of claim 49, wherein the non-human variable region is murine.
51. A polypeptide which comprises an amino acid sequence which is substantially the same as the amino acid sequence of the hypervariable region of the monoclonal antibody of claim 40 and that has the same binding specificity.
52. A nucleic acid that encodes the polypeptide of claim 51.
53. A humanized antibody or a fragment thereof comprising the polypeptide of claim 51.
54. The humanized antibody of claim 53 comprising amino acid sequences of framework and constant regions from a human antibody, and an amino acid sequence of a non-human antibody hypervariable region.
55. The humanized antibody of claim 54, wherein the amino acid sequence of the hypervariable region is murine.
56. A method of inhibiting an unwanted angiogenic condition in a mammal in need thereof comprising treating the mammal with an effective amount of a vector that expresses an immunogen that causes an immune response against a molecule that induces angiogenesis in the mammal.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US3672498A | 1998-03-06 | 1998-03-06 | |
US09/036,724 | 1998-03-06 | ||
PCT/US1999/005164 WO1999045018A1 (en) | 1998-03-06 | 1999-03-08 | Active immunization against angiogenesis-associated antigens |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2310252A1 true CA2310252A1 (en) | 1999-09-10 |
Family
ID=21890263
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002310252A Abandoned CA2310252A1 (en) | 1998-03-06 | 1999-03-08 | Active immunization against angiogenesis-associated antigens |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1032583A4 (en) |
AU (1) | AU2994599A (en) |
CA (1) | CA2310252A1 (en) |
WO (1) | WO1999045018A1 (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2364503A1 (en) * | 1999-03-11 | 2000-09-14 | Entremed, Inc. | Compositions and methods for treating cancer and hyperproliferative disorders |
FR2796073B1 (en) * | 1999-07-07 | 2003-08-29 | Centre Nat Rech Scient | ANTI-IDIOTYPIC ANTIBODIES FOR FIBROBLAST GROWTH FACTORS AND THEIR USE AS MEDICAMENTS |
US7087592B1 (en) | 1999-08-23 | 2006-08-08 | Entre Med, Inc. | Compositions comprising purified 2-methoxyestradiol and methods of producing same |
US7094410B2 (en) * | 2002-03-02 | 2006-08-22 | The Scripps Research Institute | DNA vaccine against proliferating endothelial cells and methods of use thereof |
CU23178A1 (en) * | 2002-04-15 | 2006-09-22 | Ct Ingenieria Genetica Biotech | ANTIANGIOGEN ACTIVE ACTIVE IMMUNOTHERAPY |
KR20050047518A (en) * | 2002-07-05 | 2005-05-20 | 듀크 유니버시티 | Angio-immunotherapy |
CA2646065C (en) | 2006-03-20 | 2014-01-14 | Entremed, Inc. | Disease modifying anti-arthritic activity of 2-methoxyestradiol |
WO2008006187A2 (en) * | 2006-07-12 | 2008-01-17 | Legeev, Yury V. | Protein complexes for prevention and treatment of diseases with angiogenesis disorders |
CU24637B1 (en) | 2019-12-24 | 2022-12-12 | Ct Ingenieria Genetica Biotecnologia | POLYPEPTIDES INCLUDING HUMAN VEGF-A MUTANTS WITH DISULFIDE BRIDGE RE-ARRANGEMENTS AND COMPOSITIONS CONTAINING THEM |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0006916B1 (en) * | 1977-09-28 | 1981-07-08 | National Research Development Corporation | Immunological preparations incorporating mhc antigens and their production |
US4778752A (en) * | 1983-10-11 | 1988-10-18 | Scripps Clinic And Research Foundation | Receptors specific for hapten-modified self proteins |
US5703055A (en) * | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
AU651949B2 (en) * | 1989-07-14 | 1994-08-11 | American Cyanamid Company | Cytokine and hormone carriers for conjugate vaccines |
EP0438803B1 (en) * | 1990-01-26 | 1997-03-12 | Immunomedics, Inc. | Vaccines against cancer and infectious diseases |
US5965132A (en) * | 1992-03-05 | 1999-10-12 | Board Of Regents, The University Of Texas System | Methods and compositions for targeting the vasculature of solid tumors |
US5580563A (en) * | 1992-05-01 | 1996-12-03 | Tam; James P. | Multiple antigen peptide system having adjuvant properties, vaccines prepared therefrom and methods of use thereof |
EP0666761A4 (en) * | 1992-08-31 | 1996-07-17 | Jenner Technologies | Stimulation of an antitumor t-cell response using anti-idiotypic antibodies. |
FR2742662B1 (en) * | 1995-12-21 | 1998-01-23 | Centre Nat Rech Scient | ANTI-IDIOTYPIC ANTIBODIES OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND THEIR USE AS MEDICAMENTS |
EP2057999A3 (en) * | 1995-12-22 | 2009-07-29 | Immunomedics, Inc. | Use of immunoconjugates to enhance the efficacy of multi-stage cascade boosting vaccines |
-
1999
- 1999-03-08 CA CA002310252A patent/CA2310252A1/en not_active Abandoned
- 1999-03-08 AU AU29945/99A patent/AU2994599A/en not_active Abandoned
- 1999-03-08 EP EP99911258A patent/EP1032583A4/en not_active Withdrawn
- 1999-03-08 WO PCT/US1999/005164 patent/WO1999045018A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
EP1032583A1 (en) | 2000-09-06 |
WO1999045018A1 (en) | 1999-09-10 |
AU2994599A (en) | 1999-09-20 |
EP1032583A4 (en) | 2005-02-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6215270B2 (en) | GITR binding molecules and uses thereof | |
JP5597793B2 (en) | ILT3-binding molecules and uses thereof | |
JP5372500B2 (en) | ILT3-binding molecules and uses thereof | |
US6509016B1 (en) | Monoclonal antibody 1A7 and use for the treatment of melanoma and small cell carcinoma | |
EP0876486B1 (en) | Murine monoclonal anti-idiotype antibody 11d10 an methods of use thereof | |
US8187600B2 (en) | Polypeptides capable of binding to CD64 comprising one or more heterologous T cell epitopes and their uses | |
KR20210084648A (en) | Plasmid constructs and methods of use for treating cancer | |
CA2310252A1 (en) | Active immunization against angiogenesis-associated antigens | |
US20040265274A1 (en) | Anti-tumor molecular vaccine and method of making thereof | |
US20030212027A1 (en) | Vaccine formulations and methods for immunizing an individual against shed antigen specific B cells | |
AU2002225230A1 (en) | Polypeptides capable of binding to CD64 and comprising one or more heterologous T cell epitopes, and their uses | |
JP2021526013A (en) | Anti-human LAG-3 monoclonal antibody and its applications | |
Cui et al. | The anti-tumor immune responses induced by a fusion protein of ovarian carcinoma anti-idiotypic antibody 6B11ScFv and murine GM-CSF in BALB/c mice | |
Tunheim et al. | Human receptors of innate immunity (CD14, TLR2) are promising targets for novel recombinant immunoglobulin-based vaccine candidates | |
JP3683278B2 (en) | Mouse monoclonal anti-idiotype antibody 11D10 and method of use thereof | |
EP1161147A1 (en) | Peptide mimics useful for treating disease | |
JP2023505311A (en) | antigen load | |
WO2012018260A1 (en) | Epidermal growth factor receptor targeted immune therapy | |
Ko | Dendritic cell-based approaches for the immunotherapy of malignant diseases | |
JP2001055341A (en) | Method and composition for reconstructing antigen containing many epitopes to cause immune response | |
WO1999053931A1 (en) | Tumor cells with increased immunogenicity and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Discontinued |