US20080254050A1 - Antiangiogenic active immunotherapy - Google Patents
Antiangiogenic active immunotherapy Download PDFInfo
- Publication number
- US20080254050A1 US20080254050A1 US11/787,522 US78752207A US2008254050A1 US 20080254050 A1 US20080254050 A1 US 20080254050A1 US 78752207 A US78752207 A US 78752207A US 2008254050 A1 US2008254050 A1 US 2008254050A1
- Authority
- US
- United States
- Prior art keywords
- vegf
- dna
- pmae5δ5
- tumor
- cells
- 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
- 238000009169 immunotherapy Methods 0.000 title abstract description 6
- 230000001772 anti-angiogenic effect Effects 0.000 title description 6
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 69
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 claims description 30
- 239000012634 fragment Substances 0.000 claims description 17
- 230000002163 immunogen Effects 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 6
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 3
- 241000588650 Neisseria meningitidis Species 0.000 claims description 2
- 108020001507 fusion proteins Proteins 0.000 claims description 2
- 102000037865 fusion proteins Human genes 0.000 claims description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 99
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 abstract description 81
- 201000011510 cancer Diseases 0.000 abstract description 27
- 238000000034 method Methods 0.000 abstract description 20
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 18
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 18
- 238000011282 treatment Methods 0.000 abstract description 16
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 abstract description 14
- 206010027476 Metastases Diseases 0.000 abstract description 12
- 230000009401 metastasis Effects 0.000 abstract description 11
- 230000033115 angiogenesis Effects 0.000 abstract description 10
- 206010012601 diabetes mellitus Diseases 0.000 abstract description 9
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 8
- 230000006020 chronic inflammation Effects 0.000 abstract description 8
- 210000000056 organ Anatomy 0.000 abstract description 8
- 208000010412 Glaucoma Diseases 0.000 abstract description 6
- 206010038923 Retinopathy Diseases 0.000 abstract description 6
- 201000011066 hemangioma Diseases 0.000 abstract description 6
- 208000002780 macular degeneration Diseases 0.000 abstract description 6
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 5
- 206010052779 Transplant rejections Diseases 0.000 abstract description 5
- 229920001184 polypeptide Polymers 0.000 abstract description 5
- 208000003120 Angiofibroma Diseases 0.000 abstract description 4
- 230000008578 acute process Effects 0.000 abstract description 4
- 201000003142 neovascular glaucoma Diseases 0.000 abstract description 4
- 230000001575 pathological effect Effects 0.000 abstract description 4
- 208000035473 Communicable disease Diseases 0.000 abstract description 2
- 238000002648 combination therapy Methods 0.000 abstract description 2
- 238000011125 single therapy Methods 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract 1
- 230000004048 modification Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 62
- 239000013598 vector Substances 0.000 description 46
- 108090000623 proteins and genes Proteins 0.000 description 44
- 210000004027 cell Anatomy 0.000 description 42
- 241001465754 Metazoa Species 0.000 description 39
- 241000699670 Mus sp. Species 0.000 description 38
- 241000699666 Mus <mouse, genus> Species 0.000 description 37
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 33
- 238000002649 immunization Methods 0.000 description 30
- 230000003053 immunization Effects 0.000 description 29
- 102000005962 receptors Human genes 0.000 description 24
- 108020003175 receptors Proteins 0.000 description 24
- 230000004083 survival effect Effects 0.000 description 24
- 230000014509 gene expression Effects 0.000 description 23
- 239000013642 negative control Substances 0.000 description 23
- 235000018102 proteins Nutrition 0.000 description 22
- 102000004169 proteins and genes Human genes 0.000 description 22
- 108010029485 Protein Isoforms Proteins 0.000 description 20
- 102000001708 Protein Isoforms Human genes 0.000 description 20
- 239000013612 plasmid Substances 0.000 description 20
- 229960005486 vaccine Drugs 0.000 description 20
- 108020004635 Complementary DNA Proteins 0.000 description 19
- 239000000427 antigen Substances 0.000 description 18
- 108091007433 antigens Proteins 0.000 description 18
- 102000036639 antigens Human genes 0.000 description 18
- 238000001727 in vivo Methods 0.000 description 18
- 241000700605 Viruses Species 0.000 description 17
- 238000010804 cDNA synthesis Methods 0.000 description 17
- 239000002299 complementary DNA Substances 0.000 description 17
- 210000001744 T-lymphocyte Anatomy 0.000 description 15
- 230000001225 therapeutic effect Effects 0.000 description 15
- 239000002671 adjuvant Substances 0.000 description 14
- 230000002018 overexpression Effects 0.000 description 14
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 13
- 230000000875 corresponding effect Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 102000058223 human VEGFA Human genes 0.000 description 13
- 210000003719 b-lymphocyte Anatomy 0.000 description 11
- 201000001441 melanoma Diseases 0.000 description 11
- 230000009467 reduction Effects 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 11
- 210000004698 lymphocyte Anatomy 0.000 description 10
- 206010039073 rheumatoid arthritis Diseases 0.000 description 10
- 238000002255 vaccination Methods 0.000 description 10
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 9
- 102100038232 Vascular endothelial growth factor C Human genes 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 210000004881 tumor cell Anatomy 0.000 description 9
- 208000026310 Breast neoplasm Diseases 0.000 description 8
- 238000011740 C57BL/6 mouse Methods 0.000 description 8
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 8
- 102100035194 Placenta growth factor Human genes 0.000 description 8
- 108010073925 Vascular Endothelial Growth Factor B Proteins 0.000 description 8
- 108010073919 Vascular Endothelial Growth Factor D Proteins 0.000 description 8
- 102100038217 Vascular endothelial growth factor B Human genes 0.000 description 8
- 102100038234 Vascular endothelial growth factor D Human genes 0.000 description 8
- 230000027455 binding Effects 0.000 description 8
- 230000036755 cellular response Effects 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 101001001487 Homo sapiens Phosphatidylinositol-glycan biosynthesis class F protein Proteins 0.000 description 7
- 241000124008 Mammalia Species 0.000 description 7
- 108091061960 Naked DNA Proteins 0.000 description 7
- 206010030113 Oedema Diseases 0.000 description 7
- 108091008605 VEGF receptors Proteins 0.000 description 7
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 7
- 231100000433 cytotoxic Toxicity 0.000 description 7
- 230000001472 cytotoxic effect Effects 0.000 description 7
- 210000004443 dendritic cell Anatomy 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 210000002889 endothelial cell Anatomy 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 230000004614 tumor growth Effects 0.000 description 7
- 208000007766 Kaposi sarcoma Diseases 0.000 description 6
- 201000004681 Psoriasis Diseases 0.000 description 6
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 210000000481 breast Anatomy 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 230000008348 humoral response Effects 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 210000001165 lymph node Anatomy 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000010076 replication Effects 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 239000003981 vehicle Substances 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 201000001320 Atherosclerosis Diseases 0.000 description 5
- 206010006187 Breast cancer Diseases 0.000 description 5
- 101710132601 Capsid protein Proteins 0.000 description 5
- 201000006082 Chickenpox Diseases 0.000 description 5
- 206010012689 Diabetic retinopathy Diseases 0.000 description 5
- 201000009273 Endometriosis Diseases 0.000 description 5
- 241001529936 Murinae Species 0.000 description 5
- 206010038687 Respiratory distress Diseases 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 206010046980 Varicella Diseases 0.000 description 5
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 206010003246 arthritis Diseases 0.000 description 5
- 208000006673 asthma Diseases 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 230000003308 immunostimulating effect Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000007170 pathology Effects 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 208000036487 Arthropathies Diseases 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 description 4
- 208000012659 Joint disease Diseases 0.000 description 4
- 108090000772 Neuropilin-1 Proteins 0.000 description 4
- 102000004207 Neuropilin-1 Human genes 0.000 description 4
- 108010053100 Vascular Endothelial Growth Factor Receptor-3 Proteins 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 210000003038 endothelium Anatomy 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 230000000091 immunopotentiator Effects 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 238000001325 log-rank test Methods 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000001543 one-way ANOVA Methods 0.000 description 4
- 210000001672 ovary Anatomy 0.000 description 4
- 230000001023 pro-angiogenic effect Effects 0.000 description 4
- 206010043778 thyroiditis Diseases 0.000 description 4
- 230000002103 transcriptional effect Effects 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 238000012800 visualization Methods 0.000 description 4
- BJHCYTJNPVGSBZ-YXSASFKJSA-N 1-[4-[6-amino-5-[(Z)-methoxyiminomethyl]pyrimidin-4-yl]oxy-2-chlorophenyl]-3-ethylurea Chemical compound CCNC(=O)Nc1ccc(Oc2ncnc(N)c2\C=N/OC)cc1Cl BJHCYTJNPVGSBZ-YXSASFKJSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 101000851030 Homo sapiens Vascular endothelial growth factor receptor 3 Proteins 0.000 description 3
- 101150088608 Kdr gene Proteins 0.000 description 3
- 208000008839 Kidney Neoplasms Diseases 0.000 description 3
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 3
- 102000004213 Neuropilin-2 Human genes 0.000 description 3
- 108090000770 Neuropilin-2 Proteins 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 206010060862 Prostate cancer Diseases 0.000 description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 3
- 238000011579 SCID mouse model Methods 0.000 description 3
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 3
- 239000002870 angiogenesis inducing agent Substances 0.000 description 3
- 230000006023 anti-tumor response Effects 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 210000003128 head Anatomy 0.000 description 3
- 208000006454 hepatitis Diseases 0.000 description 3
- 231100000283 hepatitis Toxicity 0.000 description 3
- 230000001506 immunosuppresive effect Effects 0.000 description 3
- 210000002751 lymph Anatomy 0.000 description 3
- 230000001926 lymphatic effect Effects 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 210000003739 neck Anatomy 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- YKPYIPVDTNNYCN-INIZCTEOSA-N prinomastat Chemical compound ONC(=O)[C@H]1C(C)(C)SCCN1S(=O)(=O)C(C=C1)=CC=C1OC1=CC=NC=C1 YKPYIPVDTNNYCN-INIZCTEOSA-N 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 210000002307 prostate Anatomy 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 210000002784 stomach Anatomy 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 206010009900 Colitis ulcerative Diseases 0.000 description 2
- 108091029430 CpG site Proteins 0.000 description 2
- 208000011231 Crohn disease Diseases 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 206010014733 Endometrial cancer Diseases 0.000 description 2
- 206010014759 Endometrial neoplasm Diseases 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 201000008808 Fibrosarcoma Diseases 0.000 description 2
- 101150048336 Flt1 gene Proteins 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 2
- 208000002125 Hemangioendothelioma Diseases 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101100395310 Homo sapiens HLA-A gene Proteins 0.000 description 2
- 102000006992 Interferon-alpha Human genes 0.000 description 2
- 108010047761 Interferon-alpha Proteins 0.000 description 2
- 206010025219 Lymphangioma Diseases 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- BKAYIFDRRZZKNF-VIFPVBQESA-N N-acetylcarnosine Chemical compound CC(=O)NCCC(=O)N[C@H](C(O)=O)CC1=CN=CN1 BKAYIFDRRZZKNF-VIFPVBQESA-N 0.000 description 2
- 208000008589 Obesity Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 208000025865 Ulcer Diseases 0.000 description 2
- 201000006704 Ulcerative Colitis Diseases 0.000 description 2
- 206010046865 Vaccinia virus infection Diseases 0.000 description 2
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000002547 anomalous effect Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 208000037976 chronic inflammation Diseases 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 210000003989 endothelium vascular Anatomy 0.000 description 2
- 210000003238 esophagus Anatomy 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 230000001497 fibrovascular Effects 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 206010027191 meningioma Diseases 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 206010061289 metastatic neoplasm Diseases 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 230000003387 muscular Effects 0.000 description 2
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 2
- 235000020824 obesity Nutrition 0.000 description 2
- 201000008482 osteoarthritis Diseases 0.000 description 2
- 201000008968 osteosarcoma Diseases 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 210000002536 stromal cell Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 231100000397 ulcer Toxicity 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 208000007089 vaccinia Diseases 0.000 description 2
- 208000006542 von Hippel-Lindau disease Diseases 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 1
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 241000710929 Alphavirus Species 0.000 description 1
- 206010073128 Anaplastic oligodendroglioma Diseases 0.000 description 1
- 108090000644 Angiozyme Proteins 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 206010071155 Autoimmune arthritis Diseases 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101000583086 Bunodosoma granuliferum Delta-actitoxin-Bgr2b Proteins 0.000 description 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 241000178270 Canarypox virus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 102000000503 Collagen Type II Human genes 0.000 description 1
- 108010041390 Collagen Type II Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 206010010099 Combined immunodeficiency Diseases 0.000 description 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 1
- 238000011238 DNA vaccination Methods 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102400001047 Endostatin Human genes 0.000 description 1
- 108010079505 Endostatins Proteins 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 208000009386 Experimental Arthritis Diseases 0.000 description 1
- 101150009958 FLT4 gene Proteins 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 101000597577 Gluconacetobacter diazotrophicus (strain ATCC 49037 / DSM 5601 / CCUG 37298 / CIP 103539 / LMG 7603 / PAl5) Outer membrane protein Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000868215 Homo sapiens CD40 ligand Proteins 0.000 description 1
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- 101000979342 Homo sapiens Nuclear factor NF-kappa-B p105 subunit Proteins 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102000000521 Immunophilins Human genes 0.000 description 1
- 108010016648 Immunophilins Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000003814 Interleukin-10 Human genes 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000003810 Interleukin-18 Human genes 0.000 description 1
- 108090000171 Interleukin-18 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 206010061523 Lip and/or oral cavity cancer Diseases 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 102100023050 Nuclear factor NF-kappa-B p105 subunit Human genes 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 201000010133 Oligodendroglioma Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102100040681 Platelet-derived growth factor C Human genes 0.000 description 1
- 102100037596 Platelet-derived growth factor subunit A Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 101150101561 TOM70 gene Proteins 0.000 description 1
- 239000012163 TRI reagent Substances 0.000 description 1
- 101710192266 Tegument protein VP22 Proteins 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 244000078912 Trichosanthes cucumerina Species 0.000 description 1
- 235000008322 Trichosanthes cucumerina Nutrition 0.000 description 1
- 102000013394 Troponin I Human genes 0.000 description 1
- 108010065729 Troponin I Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 1
- 102000016663 Vascular Endothelial Growth Factor Receptor-3 Human genes 0.000 description 1
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 208000001969 capillary hemangioma Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000008119 colloidal silica Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 208000018554 digestive system carcinoma Diseases 0.000 description 1
- GXGAKHNRMVGRPK-UHFFFAOYSA-N dimagnesium;dioxido-bis[[oxido(oxo)silyl]oxy]silane Chemical compound [Mg+2].[Mg+2].[O-][Si](=O)O[Si]([O-])([O-])O[Si]([O-])=O GXGAKHNRMVGRPK-UHFFFAOYSA-N 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000009093 first-line therapy Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000030414 genetic transfer Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 201000002222 hemangioblastoma Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 238000007489 histopathology method Methods 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 108010028930 invariant chain Proteins 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007108 local immune response Effects 0.000 description 1
- 208000037841 lung tumor Diseases 0.000 description 1
- 230000035168 lymphangiogenesis Effects 0.000 description 1
- 210000001077 lymphatic endothelium Anatomy 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 239000000391 magnesium silicate Substances 0.000 description 1
- 229940099273 magnesium trisilicate Drugs 0.000 description 1
- 229910000386 magnesium trisilicate Inorganic materials 0.000 description 1
- 235000019793 magnesium trisilicate Nutrition 0.000 description 1
- 208000006178 malignant mesothelioma Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 239000003475 metalloproteinase inhibitor Substances 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 238000004713 multireference configuration interaction Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000014399 negative regulation of angiogenesis Effects 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 101150115693 ompA gene Proteins 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 101150090944 otomp gene Proteins 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000009522 phase III clinical trial Methods 0.000 description 1
- RLZZZVKAURTHCP-UHFFFAOYSA-N phenanthrene-3,4-diol Chemical compound C1=CC=C2C3=C(O)C(O)=CC=C3C=CC2=C1 RLZZZVKAURTHCP-UHFFFAOYSA-N 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 108010017843 platelet-derived growth factor A Proteins 0.000 description 1
- 108010017992 platelet-derived growth factor C Proteins 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920002627 poly(phosphazenes) Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229950003608 prinomastat Drugs 0.000 description 1
- 208000023958 prostate neoplasm Diseases 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 210000003314 quadriceps muscle Anatomy 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 238000013515 script Methods 0.000 description 1
- 238000009094 second-line therapy Methods 0.000 description 1
- 230000018448 secretion by cell Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- FIAFUQMPZJWCLV-UHFFFAOYSA-N suramin Chemical compound OS(=O)(=O)C1=CC(S(O)(=O)=O)=C2C(NC(=O)C3=CC=C(C(=C3)NC(=O)C=3C=C(NC(=O)NC=4C=C(C=CC=4)C(=O)NC=4C(=CC=C(C=4)C(=O)NC=4C5=C(C=C(C=C5C(=CC=4)S(O)(=O)=O)S(O)(=O)=O)S(O)(=O)=O)C)C=CC=3)C)=CC=C(S(O)(=O)=O)C2=C1 FIAFUQMPZJWCLV-UHFFFAOYSA-N 0.000 description 1
- 229960005314 suramin Drugs 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 201000004595 synovitis Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 208000013076 thyroid tumor Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000005951 type IV hypersensitivity Effects 0.000 description 1
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 208000024719 uterine cervix neoplasm Diseases 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 239000002525 vasculotropin inhibitor Substances 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000000277 virosome Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
-
- 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
- A61K39/0005—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
- A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
-
- 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
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001103—Receptors for growth factors
- A61K39/001109—Vascular endothelial growth factor receptors [VEGFR]
-
- 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
- A61K39/00113—Growth factors
- A61K39/001135—Vascular endothelial growth factor [VEGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/515—Angiogenesic factors; Angiogenin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5256—Virus expressing foreign proteins
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
Definitions
- the present invention is related with the field of biotechnology and pharmaceutical industry, in particular with active immunization employing as targets molecules related with angiogenesis.
- angiogenesis The process of formation of new blood vessels from pre-existent ones is called angiogenesis. This event is widely regulated through the equilibrium of pro- and anti-angiogenic factors.
- diseases in which the course has been related with the induction of pro-angiogenic factors and the formation of new blood vessels in anomalous form are: (a) cancer (both primary tumors and their metastases), (b) acute and chronic inflammatory processes such as asthma, respiratory distress, endometriosis, atherosclerosis, and tissular edema, (c) diseases of infectious origin as the Hepatitis, and Kaposi sarcoma, (d) autoimmune diseases as diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and (e) other diseases and states as the diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma (Carmelliet P.
- a potentially attractive therapeutic procedure for many of these cases could be based on the inhibition of the activity of the pro-angiogenic factors, that stimulate the anomalous formation of blood vessels, via their neutralization, or that of their receptors, or by eliminating the sources that produces them.
- Vascular endothelium growth factors are a family of molecules that induce the formation of new vessels specifically and directly (Leung Science 246:1306, 1989; Klagsburn M, Annual Rev Physiol 33:217, 1991).
- This family includes the vascular permeability factor, also known as vascular endothelium growth factor VPF/VEGF (now denominated VEGF-A), the placenta growth factor PIGF, the platelet derived growth factors PDGF-A and PDGF-B, and other four new molecules structurally and functionally related to VEGF-A designated VEGF-B/VRF, VEGF-C/VRP, VEGD-D/FIGF, and VEGF-E. (Olofsson B et al.
- VEGF-A is a homodimeric glycoprotein formed by two 23-kDa subunits (Ferrara N, et al. Biochem Biophys Res Cons 165:198, 1989), of which five monomeric isoforms exist, derived from the differential splicing of the same RNA. These include two isoforms that remain attach to the cellular membrane (VEGF 189 and VEGF 206), and three of soluble nature (VEGF 121, VEGF 145, and VEGF 165).
- VEGF 165 is the most abundant isoform in mammal tissues, except for lung and heart, where VEGF 189 predominates (Neufeld G et al. Canc Met Rev 15:153, 1995), and in placenta, where VEGF 121 expression prevails (Shibuya M A et al. Adv Canc Res 67:281, 1995).
- VEGF-A is the most studied and characterized protein of this family, and its alteration has been described in a larger number of diseases. Its over-expression is associated to tumors of different origin and localization, and their metastasis (Grunstein J et al. Cancer Res 59:1592, 1999), chronic inflammatory processes as ulcerative colitis and Crohn's disease (Kanazawa S, et al. Am J Gastroenterol 96:822, 2001), psoriasis (Detmar M, et al. J Exp Med 180:1141, 1994), respiratory distress (Thickett D R et al. Am J Respir Crit Care Med 164:1601, 2001), atherosclerosis (Celletti F L et al.
- the cells expressing the three basic isoforms of VEGF-A: 121, 165, and 189 are the ones that grow faster in vivo; while in final stages most tumors limit expression to the VEGF 165 isoform, or, in its absence, to a combination of 121 and 189 that far from being additive, evidences a cooperation that strengthens the tumor vascular network (Grunstein J. Mol. Cell Biol 20:7282, 2000).
- PIGF PIGF, described in 1991, is not able to induce endothelial proliferation in its homodimeric form (Maglione D et al. Proc Natl Acad Sci USA 88:9267, 1991, DiSalvo J et al. J Biol Chem 270:7717, 1995). With PIGF up-regulation, and with it, of the signal transduced via VEGFR-1, the endothelial cells amplify their responses to VEGF during the change to the angiogenic phenotype associated to certain pathologies (Carmeliet P et al. Nat Med 7:575, 2001). PIGF expression has been related to the vascularization of human meningioma and glioma (Nomura M et al.
- VEGF-B has been related to breast, ovary, and kidney tumors, and to melanomas and fibrosarcomas (Sowter H M, et al. Lab. Invest. 77:607, 1997; Salven P Am. J. Pathol. 153:103, 1998, Gunningham S P et al. Cancer Res 61:3206, 2001).
- the differential expression of the VEGF-B 167 isoform in vitro has been reported in tumor cells of diverse origin (Li X, et al. Growth Factors 19:49, 2001).
- VEGF-C and VEGF-D are involved in the regulation of lymphatic vessels formation (Joukov V. et al EMBO J. 15: 290, 1996), and VEGF-C over-expression is associated to tissular edemas, to tumors of the breast, lung, head and neck, esophagus, and stomach, lymphomas, prostate cancer, and metastatic nodes (Kajita T, et al. Br J Cancer 85:255, 2001; Kitadi Y, et al Int J Cancer 93:662, 2001; Hashimoto I, et al. Br J Cancer 85:93, 2001; Kinoshita J, et al.
- the alterations on endothelial cell function induced by the molecules of the VEGF family are mediated by their binding to cell receptors of the type tyrosine kinase class 3, that so far include: VEGFR1 (Flt1), VEGFR2 (KDR/Flk1), and VEGFR3 (Flt4) (Kaipainen A J. Exp. Med. 178:2077, 1993).
- VEGFR1 Flt1
- VEGFR2 KDR/Flk1
- VEGFR3 Flt4
- Ligands identified for VEGFR1 include VEGF-A, PIGF, and VEGF-B, in decreasing order of affinity (Shibuya M Int J Biochem Cell Biol 33: 409, 2001). In endothelial cells, this receptor captures the circulating VEGF (Gille H et al EMBO J. 19:4064, 2000). The binding of VEGF-A to the VEGFR1 expressed in cells of the hematopoyetic lineage affects significantly the activation of transcriptional factor NF ⁇ B in the precursors of dendritic cells, and in B and T lymphocytes.
- VEGFR2 receptor (KDR/Flk1) mediates the biological effects of VEGF-A, and also binds VEGF-C and VEGF-D. This receptor is expressed differentially on activated endothelium and in some cell lines of tumor origin where it establishes autocrine pathways with the secreted VEGF. Apart from being involved in the already mentioned pathologies that are related with the over-expression of its ligands, the over-expression of VEGFR2 has been related with the progression of endometrial cancer (Giatromanolaki A et al, Cancer 92:2569, 2001), malignant mesothelioma (Strizzi L et al.
- Lymphangiogenesis mediated by VEGF-C and VEGF-D results from their binding to the FLT4 receptor or VEGFR3, expressed in the lymphatic endothelium.
- the over-expression of the receptor has been related to an adverse prognosis in the course of a group of pathologic entities, including: diabetic retinopathy (Smith G. Br J Opthalmol 1999 April; 83(4):486-94), chronic inflammation and ulcers (Paavonen K et al, Am J Pathol 156:1499, 2000), the establishment of metastasis in lymphatic nodes and progression of breast cancer (Gunningham S P.
- VEGFR3 is a sensitive marker of Kaposi sarcoma, type Dabska hemangioendothelioma and of cutaneous lymphangiomatosis (Folpe A L et al. Mod Pathol 13:180, 2000; Lymboussaki A et al. Am J Pathol 153:395, 1998).
- NRP1 and NRP2 are neurophilins family (NRP), and act as co-receptors for some specific isoforms of proteins of the VEGF family: VEGF-A 145 VEGF-A 165 , VEGF-B 167 and PIGF1, increasing their mitogenic capacity.
- NRP1 neurophilins family
- the expression of NRP1 has become a marker of the aggressiveness of prostate cancer, has been related to the increase of angiogenesis in melanomas, and with apoptosis escape events in breast cancer (Latil A et al. Int J Cancer 89:167, 2000; Lacal P M J Invest Dermatol 115:1000, 2000; Bachelder RE Cancer Res 61:5736, 2001).
- NRP1, KDR, and VEGF-A 165 have been related to the fibrovascular proliferation in diabetic retinopathy cases and rheumatoid arthritis (Ishida S. et al. Invest Opthalmol Vis Sci 41: 1649, 2000; Ikeda M. Et al. J Pathol 191:426, 2000).
- NRP2 is over-expressed in osteosarcomas where it promotes angiogenesis and tumor growth (Handa A et al. Int J Oncol 17:291, 2000).
- SAI specific active immunotherapy
- antigens as peptides, proteins or DNA are employed, mixed with appropriate adjuvants.
- SAI procedures pursue the stimulation of an immune response, both of the humoral (activation of B-lymphocytes), and cellular types (activation of T helper, and cytotoxic lymphocytes, and natural killer cells), associated to dendritic cell function as professional presenting cells in the MCHI and MHC II contexts (Bystryn J C, Medscape Hematology-Oncology 4:1, 2001; Parker, K C et al., J. Immunol 152:163, 1994; Nestle F O et al., Nature Medicine 7:761, 2001; Timmerman J M, Annual Review Medicine 50:507, 1999).
- SAI is a rapidly growing field of experimental and clinical research, with attractive applications, especially in oncology, where more than 60 undergoing clinical trials based in procedures of SAI are reported, which surpass at present the clinical trials based on the use of monoclonal antibodies.
- the antigens used as immunogens for SAI are selected because of their physiological relevance and difficulty of being substituted in the processes of tumor phenotypic drift (Bodey B et al., Anticancer Research 20: 2665, 2000), and because of their high specific association with the growth and evolution of tumor tissues.
- the strategy of treating cancer using SAI also considers preferably the identification of antigens expressed in different tumor types, what could increase the number of indications for the same vaccine preparation.
- examples of these are carcinoembryonic antigen (CEA), HER2-neu, human telomerase, and gangliosides (Greener M., Mol Med Today 6:257 2000; Rice J, et al. J Immunol 167:1558, 2001; Carr A et al, Melanoma Res 11:219, 2001; Murray J L, et al. Semin Oncol 27:71, 2000).
- VEGF is over-express in the tumor compartment (Ferrara, N. Curr. Top. Microbiol. Immunol. 237:1, 1999), and high levels of VEGF and its receptors have been demonstrated in the tumor-associated vasculature (Brekken R A. J Control Release 74:173, 2001).
- the stromal cells also produce VEGF in response to the stimulus of transformed cells, with the result that when tumor cells are removed, VEGF levels persist in the patients.
- the presence of VEGF and its receptors have a practical value for the establishment of prognosis and staging in cases of prostate, cervix, and breast tumors (George D J et al.
- VEGF is also within the group of soluble factors that, together with other cytokines like IL-10, TNF- ⁇ and TGF- ⁇ , (Ohm J E y Carbone D P, Immunol Res 23:263, 2001), could be implicated in the immunosuppression that characterizes cancer patients (Staveley K, et al. Proc Natl Acad Sci USA 95:1178, 1998; Lee K H, et al. J Immunol 161:4183, 1998). This immunosuppressive effect seems to be related to its binding to the Flt1 receptor (Gabrilovich D et al. Blood 92:4150, 1998).
- the present invention describes procedures of SAI in experimental tumors using molecules of the VEGF family and their receptors.
- the antitumoral effects obtained could be based in at least four different mechanisms, without discarding their possible combinations: (a) direct destruction of cancer and stromal cells producing VEGF, by cytotoxic lymphocytes, (b) damaging of endothelial cells of tumor-associated vessels due to the capture or neutralization of the circulating VEGF via antibodies, (c) direct destruction of endothelial cells that express VEGF receptors, by cytotoxic lymphocytes or complement fixing antibodies, (d) activation of a local immune response as a consequence of the capture or neutralization of circulating VEGF, and its consequent elimination of its immunosuppressive effects.
- these treatments could be used to diminish or avoid the appearance of metastasis, to reduce or eliminate primary tumors as a first or second line therapy, in combination or not with other anti-tumor agents.
- Active immunization directed to VEGF family and their receptors could also be efficient in the single or combined therapy of acute and chronic inflammatory processes (asthma, respiratory distress, endometriosis, atherosclerosis, tissular edema), infectious diseases (Hepatitis, Kaposi sarcoma), autoimmune diseases (diabetes, psoriasis, rheumatoid arthritis, thyroiditis, synovitis), diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma, among others.
- acute and chronic inflammatory processes asthma, respiratory distress, endometriosis, atherosclerosis, tissular edema
- infectious diseases Hepatitis, Kaposi sarcoma
- autoimmune diseases diabetes, psoriasis, rheumatoid arthritis, thyroiditis, synovitis
- the in vivo administration of oligonucleotide sequences encoding for proteins of the VEGF family, their receptors, co-receptors or their fragments, as well as of their polypeptidic variants induces a cellular and humoral immune response with antiangiogenic and antitumoral effect.
- Immunogens of polypeptidic nature of interest for the present invention can be isolated from their natural sources or obtained by synthesis or recombinant DNA technology. These polypeptides can also be produced fused to proteins with acknowledged adjuvant activity like p64K (R. Silva et al U.S. Pat. No. 5,286,484 y EP 0474313), or can be covalently bound to them after their individual obtainment. Other available strategy in these cases is the obtainment of the natural polypeptide, its mutated or modified variants, and their fragments, as a part of loops exposed or not in bacterial proteins like OMP1, which are part of immunostimulatory preparations, in this particular case VSSP (R. Perez et al U.S.
- HbsAg, VP2 of parvovirus, etc. bound to specific peptides that target cells or organs specialized in the induction of the immune response (CTLA4, Fc segment of the Ig, etc.), or to proteins capable of increasing biodistribution like VP22.
- the principal natural sources of the proteins of interest for the present invention are predominantly expressed in placenta, activated endothelial cells, and tumor cells.
- the mRNA of these cells or tissues is used to obtain complementary DNA (cDNA) by known methods.
- the extracted RNA is used as template for the amplification through the polymerase chain reaction (PCR) of the cDNA corresponding to the selected antigen.
- primers used are designed according to the characteristics of the vector where the cDNA is going to be inserted and to the previously reported sequences of the protein of interest.
- the coding regions are amplified in two or more overlapping fragments. These fragments include a common ligation site used to assemble the intact DNA, starting with its fragments.
- An alternative for the cloning of the antigens of interest is the selection from commercially available DNA libraries derived from human endothelium, or from tumors of this same origin.
- These mutations are made preferably in the receptor binding sites already described in the literature.
- primers are designed that cover both ends of the desired molecule, and the PCR products are used as template to obtain the mutated molecule.
- These mutated variants lack biological activity but reproduce the immunogenic properties of the selected antigen.
- the cDNA molecules obtained by the methods described earlier are administered in a proper vector, being this a virus, a plasmid, a bacterial artificial chromosome, or similar.
- the vector carries the elements needed for the adequate expression of the gene in target cells, as well as the rest of elements that allows it to be produced in the host cellular system according to its nature.
- DNA molecules of the present invention might contain one or more genes of interest, constituted by one or more nucleic acids (cDNA, gDNA, synthetic or semi-synthetic DNA, or similar) that when transcribed or translated (when appropriate) in target cells generates the products with therapeutic/vaccine value.
- the gene of the vaccine therapeutic product according to the invention is under the control of a transcriptional promoter that is functional in the target cell or the organism (mammals), as well as of a 3′ end region that contains the signals needed for termination and polyadenilation of the mRNA of the product of interest, allowing its expression.
- the promoter can be the natural promoter of the gene or a heterologous promoter transcriptionally active in the target cell.
- the promoter can be from eukaryotic or viral origin. Among eukaryotic promoters, it is possible to use any promoter or derived sequence that stimulates or represses the gene transcription, specifically or not, inducible or not, in a strong or weak manner. Additionally, the promoter region can be modified by the insertion of activators or inductor sequences, allowing the tissue-specific or predominant expression of the gene in question.
- the gene of interest can contain a signal sequence for subcellular localization, in a way that its cellular localization or secretion could be modified in the cell where it is expressed, or elsewhere, once synthesized. It can also contain a sequence encoding for a region of specific binding to a ligand specific of immune tissue, being directed to the site where the response is generated, with the obtainment of the therapeutic/vaccine effect.
- the gene of interest can be preceded by the coding sequence for the mRNA replication machinery, in a way that mRNA is amplified in the target cell, increasing the expression of said gene, and with it, of the therapeutic/vaccine effect according to the invention.
- the replication machinery in question could of alphavirus origin (Schlesinger S., Expert Opin Biol Ther. 1:177, 2001), more specifically derived from the Sindbis or Semliki viruses, or similar.
- the gene of interest is under the transcriptional control of a subgenomic promoter that allows the amplification of its mRNA in target cells, once the molecules according to the present invention have been internalized.
- the DNA vector might contain sequences that permit the replication of the molecules object of the present invention in mammalian cells. This allows an increase in the expression levels and/or of the therapeutic/vaccine effect (Collings A., Vaccine 18: 4601, 1999)
- the DNA vector can be purified using standard techniques for plasmid DNA purification. These techniques include the method of purification by cesium chloride density gradient, in the presence of ethidium bromide, or alternatively, the use of ionic exchange columns or any other exchanger or method to separate DNA molecules (Ferreira G N, et al, Trends Biotechnol. 18:380, 2000).
- the present invention includes the use of plasmidic DNA vectors, preferably those of the PAEC family of compact vectors for DNA immunization and gene therapy in humans (Herrera et al, Biochem. Biophys. Res. Commu. 279: 548, 2000).
- This family comprises vectors pAEC-K6 (Access number AJ278712), pAEC-M7 (Access number AJ278713), pAEC- ⁇ 2 (Access number AJ278714), pAEC-SPE (Access number AJ278715) and pAEC-SPT (Access number AJ278716).
- vectors contain only the essential elements for the expression of the product of interest in mammalian cells, including human cells, and a replication unit in Escherichia coli .
- the transcriptional unit is formed by the immediate early promoter of human cytomegalovirus (CMV), a versatile multicloning site for the insertion of the product of interest, and the sequences for transcriptional termination and polyadenilation derived from simian virus 40 (SV40).
- CMV human cytomegalovirus
- SV40 simian virus 40
- the vector contains the gene for kanamycin resistance (Tn903), and a pUC19 replication origin (ColE1), in order to guarantee a high copy number and the selection of the bacteria that bear the plasmid of interest.
- the present invention includes the use of plasmidic DNA vectors, preferably those of the PMAE family of compact vectors for DNA immunization in humans. These contain the same functional elements in bacteria as PAEC series, as well as the CMV immediate early promoter and the multicloning site. Additionally, they bear a synthetic intron and a synthetic sequence for transcription termination and polyadenilation, derived from rabbit ⁇ -globin. It has been reported that with sequences similar to the latter it is possible to obtain higher expression levels of the cloned gene (Norman J A et al, Vaccine 15: 801, 1997).
- the vectors of this series include consecutive repetitions of immunostimulatory sequences (CpG motives), that stimulate innate immune system in both mice and humans, with the consequent activation of a humoral and cellular response against the molecule of interest (Krieg A M, Vaccine 19:618, 2001).
- CpG motives immunostimulatory sequences
- the immunization with recombinant virus produces a potent cytotoxic cellular response in the hosts.
- recombinant virus adenovirus, adeno-associated, vaccinia, chickenpox virus, canarypox virus, among others
- vaccinia adenovirus
- chickenpox virus adeno-associated, vaccinia
- canarypox virus canarypox virus, among others
- chickenpox virus and the pFP67xgpt vector are preferably used.
- the pFP67xgpt vector is used to clone genes under a strong early/late promoter of synthetic nature between the open reading frames 6 and 7 of a fragment of 11.2 kB BamHI of the chickenpox virus FP9.
- This plasmid also contains the Ecogpt controlled by the vaccinia promoter p7.5K, which is used to identify recombinant virus.
- cDNA molecules obtained as previously described are cloned in vectors for expression in virus, yeast, phage, plants, or superior cells, in order to obtain the protein variants of the antigens, after their sequence has been verified by the traditional methods of automatic sequencing.
- vectors for expression have been described and used for the obtainment of recombinant proteins. These vectors contain, at least, a sequence that controls the expression operatively linked to the sequence of the DNA or fragment to be expressed.
- sequences useful for the control of expression are: the systems lac, trp, tac, and trc, the promoter regions and the principal operator of lambda phage, the controller region of the surface protein fd, the glycolytic promoters of yeast (for example, the 3-phosphoglicerate kinase), the promoters of yeast acid phosphatase (for example, Pho5), the yeast promoters for the mating alpha factor, and the promoters derived from polyoma, adenovirus, retrovirus, simian virus (for example, the early/late promoters of SV40), and other known sequences that regulate the expression of genes in prokaryotic and eukaryotic cells, their viruses, and their combinations.
- the hosts used for the replication of these vectors and the obtainment of the recombinant proteins object of the present invention include prokaryotic and eukaryotic cells.
- the prokaryotic comprise E. coli (DHI, MRCI, HB101, W3110, SG-936, X1776, X2282, DH5a), Pseudomonas, Bacillus subtilis, Streptomices , and others.
- the eukaryotic cells include yeast and fungi, insects, animal cells (for example, COS-7 and CHO), human, and plant cells, and tissue cultures, among others. After the expression in the system of choice in an adequate media, the polypeptides or peptides can be isolated by known procedures.
- the DNA or protein vaccines can be combined with immunopotentiators already described like: mineral salts (ex., Aluminum hydroxide, aluminum phosphate, calcium phosphate); immunostimulators like: cytokines (ex., IL-2, IL-12, GM-CSF, IFN- ⁇ , IFN- ⁇ , IL-18), molecules (ex., CD40, CD154, invariant chain of MHC type I, LFA3); saponins (ex., QS21), MDP derivatives, CpG oligos, LPS, MPL and polyphosphazenes; lipidic particles: like: emulsions (ex., Freund, SAF, MF59), liposomes, virosomes, iscoms, co-chelators; microparticular adjuvants like PLG microparticles
- the vaccine preparations of the present invention are administered to a mammal, preferably a human, in a dose pharmaceutically acceptable, by the following routes: mucosal, subcutaneous, intramuscular, peritoneal, intra-lymphatic, topic, and by inhalation, among others.
- routes mucosal, subcutaneous, intramuscular, peritoneal, intra-lymphatic, topic, and by inhalation, among others.
- These could be administered on the tissue interstitial space, including: muscle, skin, brain, lung, liver, bone marrow, spleen, thymus, heart, lymph nodes, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testicles, ovary, uterus, rectum, eye, glands, and connective tissue.
- vectors for oligonucleotide transfer their expression is preferably directed to somatic differentiated cells, though they may be directed to non-differentiated or less differentiated cells like skin fibroblasts and blood pl
- the doses of the immunogen could be administered in pharmaceutically accepted vehicles without toxicity or therapeutic effects.
- these vehicles include: ionic exchangers, alumina, aluminum esthearates, lecitine, seric proteins like albumin, buffer solutions, like phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated fatty acids of plant origin, water, salts, or electrolites, like protamine sulphate, di-sodic hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pirrolidone, substances base on cellulose and polyethylene glycol.
- phosphate buffers as vehicles of the vaccine preparations are used.
- proteins and peptides can be conjugated in covalent or non-covalent manner to molecules known as carriers that act like adjuvants.
- these molecules are: KLH, p64K, OPC (Musacchio A et al, Vaccine 19; 3692, 2001), and VSSP.
- the combination of naked DNA, viral vectors, and protein immunogens is an alternative also included within the scope of the present invention.
- plasmid DNA administration allows the generation of formulations with one or more molecules of interest in the vaccine preparation.
- molecules according to the present invention can be administered in vaccine schedules through the combination of different types of vectors (variant of induction re-stimulation, with DNA, proteins, viral vectors)
- DNA vectors could be directly administered to the patient, or host cells can be in vivo or ex vivo modified with these vectors. This last strategy can be combined with the insertion by site-specific recombination or the immunization by somatic transgenesis that directs the vector expression to specific cells. Furthermore, bacterial hosts of DNA vectors could be used as their vehicles of transfer in vivo.
- the molecules carrying the genes according to the present invention could be used in the form of naked DNA, or in combination with different vectors: chemical/biochemical/biologic, natural/synthetic or recombinant.
- These molecules can be coupled or combined with cationic peptides, compacting molecules (ex. PEG, PEI), nuclear localization peptides (NLP), etc.
- cationic peptides such as a polypeptide that can be administered to administering molecules.
- compacting molecules ex. PEG, PEI
- NLP nuclear localization peptides
- These could be administered also together with cations capable of forming DNA precipitates, as a part of liposomal preparations to which the molecules have been added previously to the membrane fusion, and in synthetic vectors of lipid nature, or formed by cationic polymers (ex. DOGS or DOTMA).
- DNA molecules carrying the therapeutic/vaccine genes according to the invention could be used for the genetic transfer to cells using physical methods of transfer, like particles bombardment, electroporation (in vitro, in vivo or ex vivo), or directly in vivo by topic application, inhalation by particulation, etc.
- the live vectors include adenoviral particles or the same hosts where the molecules according to the present invention have been generated.
- the doses of polypeptides and/or oligonucleotides to be used can be established according to different parameters, in particular depending on the gene or protein administered as an immunogen, the route of administration, the pathology to be treated, the period of treatment, and in the case of using oligonucleotides, of the vector used for immunization.
- a change in dose schedule or administration route different to those described in the following examples, do not separate from the principle or precept of the present invention, being possible to achieve an optimization of the immunization schemes to obtain a better response.
- the present invention has advantages over passive immunotherapy, which is in advanced phases of clinical trials using the same molecules as targets.
- the immunization in comparison with passive transfer of immunity through the administration of monoclonal antibodies (ex. Anti-VEGF), the immunization, be it with the protein or the oligonucleotide, has the advantage of inducing the endogen production of antibodies and in addition the proliferation and expansion of specific cytotoxic CD8+ lymphocytes.
- the present invention has advantages over the therapeutic strategies directed to block VEGF-VEGFRs system, mainly because these strategies only diminish the levels of circulating VEGF or block KDR.
- the strategy proposed apart from achieving the mentioned effects, also destroys the source of VEGF (that is, the tumor cells and associated stroma) and/or the cells expressing their receptors (tumor endothelium and some tumor cells). Previous work done in this area only describe a humoral response as a principal component of the observed effect.
- the examples show that, besides from the humoral specific response, the vaccine compositions are able to elicit a CD8+ cellular response that cooperates with the humoral response; and that in the tumor context, the combination of both are relevant to obtain an ant-tumor effect, the previous observed in example 9.
- cytotoxic cellular response is mediated by the recognition of some of the peptides that appear in Tables 1 and 2.
- some peptidic segments appear, that could be relevant in the cellular response directed to selected targets in VEGF family, its receptors and co-receptors.
- This information was obtained through computer analyses on public databases from NIH and Heidelberg Institute (http://bimas.dcrt.nih.gov/molbio/hla_bind, and www.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm) using BIMAS and SYFPHEITI software, respectively.
- the peptides marked and other sequences derived from the antigens of interest could be used for the active immunotherapy of the already described pathologies, as a single or combined treatment, and as part or not of molecules with adjuvant capacities. These peptides can also be used in their oligonucleotide variants with vaccine purposes.
- the methods to inhibit angiogenesis and the pathologic conditions associated to this event comprise the administration of an effective amount of the DNA or protein of some of the molecules described in this invention, by any of the routes, and with the use of some of the previously described immunopotentiators or adjuvants, to a mammal.
- This mammal is preferably a human.
- a non-reversible and unregulated increase of angiogenesis has been related to a wide group of diseases.
- the system that comprises the VEGF family, its receptors and co-receptors is over-expressed in many of these pathological conditions, as it has been described before.
- the therapeutic strategies proposed by the present invention result effective in the treatment of: (a) cancer (both primary tumors and their metastasis), (b) acute and chronic inflammatory processes like asthma, respiratory distress, endometriosis, atherosclerosis, and tissular edema, (c) diseases of infectious origin like Hepatitis and Kaposi sarcoma, (d) autoimmune diseases like diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and (e) other diseases and states such as diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma and angiofibroma.
- cancer both primary tumors and their metastasis
- acute and chronic inflammatory processes like asthma, respiratory distress, endometriosis, atherosclerosis, and tissular edema
- diseases of infectious origin like Hepatitis and Kaposi sarcoma
- autoimmune diseases like diabetes, psoriasis,
- tumors that can be treated with the proposed strategies include epidermoid tumors, squamous tumors like those of the head and neck, and colorectal, prostate, breast, lung (including small and non-small cells), pancreas, thyroid, ovary, and liver tumors.
- Kaposi sarcoma central nervous system neoplasia (neuroblastoma, capillary hemangioma, meningioma and brain metastasis), melanomas, renal and gastrointestinal carcinomas, rhabdomyosarcoma, glioblastoma and leiomiosarcoma.
- VEGF-A and/or their receptors VEGFR-1 and VEGFR-2 as immunogen is useful for the treatment of: tumors of different origins and localizations and their metastasis, of hemangioma, of endometriosis, of tissue edemas, of chronic inflammatory processes like ulcerative colitis and Crohn's disease, of, atherosclerosis, of rheumatoid arthritis and osteoarthritis, of inflammatory arthropathies, psoriasis, respiratory distress, asthma, thyroidits, of diabetic and newborn retinopathies, macular degeneration, and glaucoma, of the autosomic VHL disease, of obesity, and of the rejection of some organ transplants.
- a response vs PIGF is useful in cases of rheumatoid arthritis and in general for the treatment of primary inflammatory arthropathies.
- VEGF-B its use as immunogen results useful in cases of breast, ovary, and kidney tumors, and for melanoma and fibrosarcoma.
- Immunization with VEGF-D can be used specifically for the treatment of lymphatic node metastasis.
- NRP1 and NRP2 co-receptors in mammal immunization results useful for the treatment, in particular, of fibrovascular proliferation in prostate cancer, melanoma, osteosarcoma, breast cancer metastasis, diabetic retinopathy, and rheumatoid arthritis.
- the use of two or more of the immunogens proposed in the present invention provides an especially efficient treatment for the inhibition of angiogenesis and tumor growth.
- These immunogens can be administered in an individual manner or by pairs using bi-cistronic vectors by the already mentioned pathways.
- vaccine compositions of the invention can be used together with, or in sequential manner, with drugs or chemotherapeutic agents, that offer a benefit to the condition under treatment.
- VEGF and its receptor are involved in the process of maturation of dendritic cells and act on B and T lymphocytes precursors.
- Example 10 demonstrates that the proposed therapeutic strategy, apart from diminishing the levels of VEGF in sera also contributes to the normalization of the proportions of B and T lymphocytes, and of mature dendritic cells. This effect favors the presentation of tumor antigens within the MHC I context, improving the quality and intensity of the immune anti-tumor response directed not only to the immunogen, but also to the other tumor-associated, tumor-specific, and over-expressed antigens, in the tumor context.
- VEGF isoforms were cloned applying the polymerase chain reaction (PCR) using as template a cDNA obtained from a previous isolation of mRNA of CaSki cell line (ATCC CRL 1550), according to the manufacturer instructions (Perkin-Elmer), and utilizing primers SEQ ID1 and SEQ ID2. Bands corresponding to the amplification products of VEGF isoforms 121, 165 and 189 were extracted from 2% agarose gels. After band digestion with endonucleases BamHI and EcoRI, the cDNAs from the VEGF isoforms were purified and cloned independently in the PAEC ⁇ 2 vector (proprietary vector of CIGB).
- PAEC ⁇ 2 vector proprietary vector of CIGB
- Resulting plasmids were sequenced and determined to have no mutations with respect to the aminoacid sequences reported by the EMBL (www.embl-heidelberg.de) for the cloned isoforms.
- the cDNA corresponding to VEGF isoforms were subsequently cloned KpnI/EcoRV on the pMAE5 ⁇ 5 vector, that among other characteristics differs from pAEC ⁇ 2 by the presence of 5 immunostimulatory CpG sites.
- VEGF KDR( ⁇ ) vascular endothelial growth factor receptor
- the fragments thus amplified were purified as referred, and were used in equimolar concentrations as a template for a fusion PCR using the primers corresponding to sequences SEQ ID7 and SEQ ID8.
- Resultant cDNA containing the mutation was digested BamHI/EcoRI, and was purified, and cloned in pAEC ⁇ 2 vector.
- the mutations introduced were checked by sequencing, and the DNA corresponding to VEGF KDR( ⁇ ) was subcloned KpnI/EcoRV in pMAE5 ⁇ 5 vector resulting in pMAE5 ⁇ 5 VEGF KDR( ⁇ ) .
- Plasmids used both in transfection and in animal vaccination were purified in endotoxin-free conditions, as described by Whalen R. et al. (Whalen R G y Davis H L. Clin Immunol Immunopathol 75:1, 1995). Briefly, DNA was purified using QIAGEN Endo-free systems following the manufacturer instructions, and the DNA was furthermore submitted to a second precipitation. Finally, DNA was dissolved in endotoxin-free PBS (SIGMA, USA) to a final concentration of 4 mg/mL.
- SIGMA endotoxin-free PBS
- the primers used correspond to sequences SEQ ID9 and SEQ ID10.
- the cDNA coding for 1-3 domains of KDR was purified, and cloned in pAEC ⁇ 2 vector. Clones positive by restriction analysis were verified by sequencing of the corresponding DNA.
- the cDNA corresponding to KDR 1-3 was then subcloned KpnI/EcoRV in the already described pMAE5 ⁇ 5 vector (pMAE5 ⁇ 5 KDR1-3).
- Chickenpox virus were replicated in chicken embryo fibroblasts (CEF), in DMEM medium supplemented with 2% of fetal bovine serum (FBS).
- the pFP67xgpt KDR C was transfected using Lipofectin (Gibco BRL, Grand Island, USA) in CEF previously infected with the attenuated strain FP9. After 24 hours, fresh medium was added and cells were cultured for other 3 to 4 days. After this time, cells were frozen-thawed three times.
- Recombinant viruses expressing the gene coding for the Ecogpt enzyme were purified in selective media with mycophenolic acid (25 ⁇ g/mL), xantine (250 ⁇ g/mL), and hypoxantine (15 ⁇ g/mL) (MXH). The correct inclusion of the gene in recombinant viruses was checked by PCR. Recombinant viruses were denominated FPKDRgpt and non-recombinants used as negative control FP.
- pMAE5 ⁇ 5-VEGF 121 (10 and 50 ⁇ g/mouse) in PBS pH 7.2 2.
- pMAE5 ⁇ 5-VEGF 165 (10 and 50 ⁇ g/mouse) in PBS pH 7.2 3.
- pMAE5 ⁇ 5-VEGF 189 (10 and 50 ⁇ g/mouse) in PBS pH 7.2 4.
- pMAE5 ⁇ 5-VEGF KDR( ⁇ ) (10 and 50 ⁇ g/mouse) in PBS pH 7.2 5.
- pMAE5 ⁇ 5-KDR 1-3 (10 and 50 ⁇ g/mouse) in PBS pH 7.2 6.
- pMAE5 KDR C (10 and 50 ⁇ g/mouse) in PBS pH 7.2 7.
- FPKDRgpt (2.5*10 7 cfu) in PBS pH 7.2 8.
- PBS pH 7.2 negative control
- filters were re-hybridized with the cDNA corresponding to a constitutive gene: the gliceraldehyde 3-phosphate dehydrogenase (GAPDH).
- GPDH gliceraldehyde 3-phosphate dehydrogenase
- mice Groups of 10 C57BL/6 mice were vaccinated or not with the following variants:
- mice were immunized by intramuscular injection (im.) in the rear left foot with a total volume of 50 ⁇ l. All the animals were re-immunized 15 days later using the original immunization regime.
- the tumor challenge was developed thirty days after the last immunization, by a subcutaneous (sc.) injection of 10 4 cells of B16-F10 melanoma (ATCC, CRL-6475) in the right ventral zone of every animal. Tumor growth was monitored with three weekly measurements until animals started to die.
- mice immunized with the pMAE5 ⁇ 5-KDR 1-3 plasmid a reduction of tumor size was evidenced at doses of 50 and 100 ⁇ g of DNA/mouse, significantly lower with respect to the negative control (Table 3).
- a survival analysis at day 33 revealed a significant increment (with respect to the negative control) of this parameter for the animals immunized with the said DNA doses of 50 and 100 ⁇ g per mouse, with respect to the un-immunized mice (group PBS pH7.2).
- pMAE5 ⁇ 5-KDR C Table 3
- a significant reduction of tumor volume was observed at the four doses used, with an increment in survival for doses from 100 to 10 ⁇ g/animal.
- the use of viral vectors reduced the volume and increased survival in the condition used for the FPKDRgpt construction (Table 3), in comparison to the respective negative control (group of mice immunized with the vector without insert FPgpt).
- mice C57BL/6 were vaccinated or not with the following variants:
- pAEC ⁇ 2-VEGF 121 (1, 10, 50 and 100 ⁇ g/mouse) in PBS pH 7.2 2.
- pMAE5 ⁇ 5-VEGF 121 (1, 10, 50 and 100 ⁇ g/mouse) in PBS pH 7.2 3.
- pMAE5 ⁇ 5-VEGF 165 (1, 10, 50 and 100 ⁇ g/mouse) in PBS pH 7.2 4.
- pMAE5 ⁇ 5-VEGF 189 (1, 10, 50 and 100 ⁇ g/mouse) in PBS pH 7.2 5.
- pMAE5 ⁇ 5 VEGF KDR( ⁇ ) (1, 10, 50 and 100 ⁇ g/mouse) in PBS pH 7.2 6.
- PBS pH 7.2 negative control
- mice were immunized by im. injection in the rear left foot with a total volume of 50 ⁇ l. All the animals were re-immunized 15 days later using the original immunization regime.
- the tumor challenge was developed thirty days after the last immunization, by a subcutaneous injection of 10 4 cells of B16-F10 melanoma (ATCC, CRL-6475) in the right ventral zone of every animal. Tumor growth was monitored with three weekly measurements until animals started to die.
- a survival analysis on day 43 evidenced a significant increase (with respect to the negative control) of the animals immunized with the variants pMAE5 ⁇ 5-VEGF 121 , pMAE5 ⁇ 5-VEGF 165 , pMAE5 ⁇ 5-VEGF 189 , and pMAE5 ⁇ 5 VEGF KDR( ⁇ ) , at doses of 50 and 100 ⁇ g per animal (Table 4).
- mice were vaccinated or not with the following variants:
- immunization (day 0) was by im. route in the rear left foot with a total volume of 50 ⁇ l. All the animals were re-immunized 15 days later using the original immunization regime.
- Group Incidence day 40 Incidence day 55 1 20/8 (40%) 20/9 (45%) 2 20/6 (30%) 20/12 (60%) 3 20/10 (50%) 20/14 (70%)
- mice Groups of 15 mice were vaccinated or not with the following variants:
- pMAE5 ⁇ 5-VEGF 121 (50 ⁇ g of DNA/mouse) in PBS pH 7.2 2.
- pMAE5 ⁇ 5-KDR 1-3 (50 ⁇ g of DNA/mouse) in PBS pH 7.2 3.
- pMAE5 KDR C (50 ⁇ g/mouse) in PBS pH 7.2 4.
- PBS pH 7.2 (Negative control)
- C57BI/6 mice were immunized by im. injection in the rear left foot with a total volume of 50 ⁇ l. All the animals were re-immunized 15 days later using the original immunization regime. Thirty days after the last immunization, the in vivo angiogenesis was evaluated in the animals using matrigel as described by Coughlin M C et al. (Coughlin M C et al. J. Clin. Invest. 101:1441, 1998). The animals previously vaccinated were divided in groups of 5 and injected subcutaneously in the abdominal middle line with 500 ⁇ l of matrigel (Becton Dickinson and Co., Franklin Lakes, N.J., USA) containing:
- VEGF 50 ng/mL, Heparin 50 U/mL 1. VEGF 50 ng/mL, Heparin 50 U/mL 2. 10 5 cells of B16-F10 melanoma
- Opc protein from the outer membrane of Neisseria meningitidis was purified according to the report of Musacchio et al. (Musacchio A et al. Vaccine, 67:751, 1997). 50 ⁇ g/mL of pMAE5 ⁇ 5-VEGF 121 were added to 10 ⁇ g/mL of Opc with gentle shaking at acid pH. The resulting complex was extensively dialyzed overnight in endo-free PBS pH 7.2 (Sigma). The level of Opc protein-plasmid DNA association (Opc-pMAE5 ⁇ 5-VEGF 121 ) was checked by DNA visualization using 1% agarose gel. More than 50% of the plasmid DNA was associated to the Opc protein.
- VSSP Very small particles coming from complex of outer membrane proteins (OMPC) of Neisseria meningitides, supplied by the Center of Molecular Immunology (R. Perez et al. U.S. Pat. Nos. 5,788,985, and 6,149,921), were used for combination with the plasmid DNA of interest.
- VSSP (1 mg) were incubated with 5 mg of pMAE5 ⁇ 5-VEGF 121 overnight with gentle agitation. The resulting material was extensively dialyzed in endo-free PBS pH 7.2 (Sigma).
- the level of VSSP-plasmid DNA association (VSSP-pMAE5 ⁇ 5-VEGF 121 ) was checked by DNA visualization using 1% agarose gel. More than 50% of the plasmid DNA was associated to the VSSP particles.
- the Hepatitis C and Hepatitis B core particulated antigens were produced according to a previous report (Lorenzo L J et al., Biochem Biophys Res Commun 281:962, 2001).
- One mg of the antigens were mixed with 5 mg of the plasmid in an overnight incubation.
- the levels of HCcAg or HBcAg-plasmid DNA association were checked by DNA visualization using 1% agarose gel. More than 50% of the DNA was associated to the antigenic particle, in each case.
- mice Groups of 10 C57BL6 mice were vaccinated or not with the following variants:
- Opc-pMAE5 ⁇ 5-VEGF 121 (1, 10 and 50 ⁇ g of DNA/mouse)
- mice Groups 10 C57BL6 mice were vaccinated or not with the following variants:
- VEGF165 (20 ⁇ g/mouse) with Complete and Incomplete Freund adjuvant Complete and Incomplete Freund adjuvant (negative control)
- VEGF 165 antigen was obtained from a commercial source (SIGMA) with more than 97% purity. Mice were immunized by the intraperitoneal route using Complete Freund's adjuvant (Sigma) with re-immunizations in days 15 and 30 by the same route but using Incomplete Freund's adjuvant. Tumor challenge, and measurements of tumor volume were similar to those described in the previous example.
- C57BL6 mice were immunized or not with doses of 50 ⁇ g of pMAE5 ⁇ 5-VEGF 121 DNA/mouse using the procedures described in the example 5. Mice were sacrificed at 45 days after first immunization. CD8+, CD4+ and B-lymphocytes of these mice were separated using magnetic beads (Dynabeads, USA), according to the manufacturer instructions.
- mice Groups of 10 six-week old C57BL6 SCID mice were reconstituted with the following combinations of the previously extracted lymphocytes.
- Group 1 CD8+ T-lymphocytes and CD4+ T-lymphocytes from mice immunized with pMAE5 ⁇ 5-VEGF 121 DNA. B-lymphocytes were not reconstituted.
- Group 2 B-lymphocytes and CD4+ T-lymphocytes from immunized mice, and CD8+ T-lymphocytes from non-immunized mice.
- Group 3 B-lymphocytes, CD8+ T-lymphocytes and CD4+ T-lymphocytes from immunized mice, as a positive control of the experiment.
- Group 4 B-lymphocytes, CD8+ T-lymphocytes, and CD4+ T-lymphocytes from non-immunized mice, as a negative control of the experiment.
- Reconstituted SCID mice were challenged sc. with 10 4 B16-F10 melanoma cells. Tumor growth was monitored by three weekly measurements until mice start to die. Anti-VEGF antibody levels were analyzed through a laboratory ELISA. 96-well plates were incubated overnight with a 0.5 ⁇ g/ml solution of VEGF165 (Sigma). The wells were blocked with PBS-BSA 1% (BDH, UK) solution, and later incubated with serial dilutions of the animal sera. After washing with PBS-Tween 0.05%, a commercially available polyclonal anti mouse IgG (Sigma, A0168) was added. The signal was amplified in the presence of the commercial substrate ortho-phenilene-diamine (OPD, Sigma).
- OPD ortho-phenilene-diamine
- Table 7 reflects the results of tumor volume (Day 24) and survival (Day 40) of the groups of mice submitted to tumor challenge. Beginning on the day 15 after reconstitution, the animals of the groups 1 to 3 experienced a reduction in tumor size as compared to group 4, reconstituted with lymphocytes from non-immunized mice. Thus, the effect that provokes the immune system in the immunized mice, that allows the reduction in tumor size, is related to humoral and cellular responses, being the last one of the cytotoxic type (CTL), due to the absence of anti-VEGF antibodies in group 1. Nevertheless, in the experimental conditions used survival only increased in group 3 (B and T lymphocytes of immunized mice), as compared to the rest of the groups (Table 7).
- CTL cytotoxic type
- Tumor volume is reported as mean ⁇ standard deviation (SD) of the measures performed on the animals of each group, statistical comparisons were performed using one-way ANOVA and a Bonferroni post-test. In the case of survival, the reported statistical significance was obtained using the log-rank test to compare each group with respect to the control group, in the indicated day.
- Statistical signification is indicated as (ns) p ⁇ 0.05 non-significant; (*) p ⁇ 0.05; (**) p ⁇ 0.01; and (***) p ⁇ 0.001.
- mice Groups of 15 C57BL6 female mice were injected by im. route with the following variants:
- mice were immunized by im. injection in the rear left foot with a total volume of 50 ⁇ l. All the animals were re-immunized 15 days later using the original immunization regime. Thirty days after the last immunization 5 randomly selected animals from each group were sacrificed to analyze the immunological state of the immunized and control animals, as well as the toxicity of vaccination on organs and tissues, through macroscopic and histological evaluations.
- Remaining animals of each group received a sc injection of 10 4 melanoma B16-F10 cells in the right ventral zone. At 15 and 30 days after tumor cells injection, 5 mice per group were sacrificed and evaluated as previously described.
- Immunological evaluation consisted of: (1) evaluation of murine VEGF levels in serum; (2) cellular content of T and B lymphocytes, as well as the degree of maturity of dendritic cells in spleen, and in brachial axillary and inguinal lymph nodes.
Abstract
Application of oligonucleotide and polypeptide sequences of molecules of the family of the vascular permeability factor (VPF), their receptors, and co-receptors, as well as their modifications, in the active immunotherapy of pathologic entities in which course is associated to the increase of angiogenesis.
These procedures can be employed in the single or combined therapy for the treatment of cancer and its metastasis, acute and chronic inflammatory processes, infectious diseases, autoimmune diseases, diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma, among others.
Description
- The present invention is related with the field of biotechnology and pharmaceutical industry, in particular with active immunization employing as targets molecules related with angiogenesis.
- The process of formation of new blood vessels from pre-existent ones is called angiogenesis. This event is widely regulated through the equilibrium of pro- and anti-angiogenic factors. Among the diseases in which the course has been related with the induction of pro-angiogenic factors and the formation of new blood vessels in anomalous form are: (a) cancer (both primary tumors and their metastases), (b) acute and chronic inflammatory processes such as asthma, respiratory distress, endometriosis, atherosclerosis, and tissular edema, (c) diseases of infectious origin as the Hepatitis, and Kaposi sarcoma, (d) autoimmune diseases as diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and (e) other diseases and states as the diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma (Carmelliet P. y Jain R K. Nature 407:249, 2000; Kuwano M, et al. Intern Med 40:565, 2001). A potentially attractive therapeutic procedure for many of these cases could be based on the inhibition of the activity of the pro-angiogenic factors, that stimulate the anomalous formation of blood vessels, via their neutralization, or that of their receptors, or by eliminating the sources that produces them.
- Vascular endothelium growth factors are a family of molecules that induce the formation of new vessels specifically and directly (Leung Science 246:1306, 1989; Klagsburn M, Annual Rev Physiol 33:217, 1991). This family includes the vascular permeability factor, also known as vascular endothelium growth factor VPF/VEGF (now denominated VEGF-A), the placenta growth factor PIGF, the platelet derived growth factors PDGF-A and PDGF-B, and other four new molecules structurally and functionally related to VEGF-A designated VEGF-B/VRF, VEGF-C/VRP, VEGD-D/FIGF, and VEGF-E. (Olofsson B et al. PNAS USA 13:2576, 1996; Joukov V et al. EMBO J 15:290, 1996; Yamada Y et al. Genomics 42:483, 1997; Ogawa S et al. J Biol Chem 273:31273, 1998).
- VEGF-A is a homodimeric glycoprotein formed by two 23-kDa subunits (Ferrara N, et al. Biochem Biophys Res Comun 165:198, 1989), of which five monomeric isoforms exist, derived from the differential splicing of the same RNA. These include two isoforms that remain attach to the cellular membrane (VEGF 189 and VEGF 206), and three of soluble nature (VEGF 121, VEGF 145, and VEGF 165). VEGF 165 is the most abundant isoform in mammal tissues, except for lung and heart, where VEGF 189 predominates (Neufeld G et al. Canc Met Rev 15:153, 1995), and in placenta, where VEGF 121 expression prevails (Shibuya M A et al. Adv Canc Res 67:281, 1995).
- VEGF-A is the most studied and characterized protein of this family, and its alteration has been described in a larger number of diseases. Its over-expression is associated to tumors of different origin and localization, and their metastasis (Grunstein J et al. Cancer Res 59:1592, 1999), chronic inflammatory processes as ulcerative colitis and Crohn's disease (Kanazawa S, et al. Am J Gastroenterol 96:822, 2001), psoriasis (Detmar M, et al. J Exp Med 180:1141, 1994), respiratory distress (Thickett D R et al. Am J Respir Crit Care Med 164:1601, 2001), atherosclerosis (Celletti F L et al. Nat Med 7:425, 2001; Couffinhal T et al. Am J Pathol 150:1653, 1997), endometriosis (McLaren J. Hum Reprod Update 6:45, 200), asthma (Hoshino M, et al. J Allergy Clin Immunol 107:295, 2001), rheumatoid arthritis and osteoarthritis (Pufe T et al. J Rheumatol 28:1482, 2001), thyroiditis (Nagura S et al. Hum Pathol 32:10, 2001), diabetic and newborn retinopathies (Murata T et al. Lab Invest 74:819, 1996; Reynolds J D. Paediatr Drugs 3:263, 2001), macular degeneration and glaucoma (Wells J A et al. Br J Opthalmol 80:363, 1996; Tripathi R C et al. Opthalmology 105:232, 1998), tissular edema (Kaner R J et al Am J Respir Cell Mol Biol. 22:640 2000; Ferrara N Endocrinol Rev 13:18, 1992), obesity (Tonello C et al. FEBS Left 442:167, 1999), hemangiomas (Wizigmann S y Plate K H Histol Histopathol 11:1049, 1996), in the synovial fluid of patients with inflammatory arthropathies (Bottomley M J et al Clin Exp Immunol 119:182, 2000), and associated to transplant rejection (Vasir B, et al. Transplantation 71:924, 2001). In the particular case of tumors, the cells expressing the three basic isoforms of VEGF-A: 121, 165, and 189, are the ones that grow faster in vivo; while in final stages most tumors limit expression to the VEGF 165 isoform, or, in its absence, to a combination of 121 and 189 that far from being additive, evidences a cooperation that strengthens the tumor vascular network (Grunstein J. Mol. Cell Biol 20:7282, 2000).
- PIGF, described in 1991, is not able to induce endothelial proliferation in its homodimeric form (Maglione D et al. Proc Natl Acad Sci USA 88:9267, 1991, DiSalvo J et al. J Biol Chem 270:7717, 1995). With PIGF up-regulation, and with it, of the signal transduced via VEGFR-1, the endothelial cells amplify their responses to VEGF during the change to the angiogenic phenotype associated to certain pathologies (Carmeliet P et al. Nat Med 7:575, 2001). PIGF expression has been related to the vascularization of human meningioma and glioma (Nomura M et al. J Neurooncol 40:123, 1998). This molecule forms heterodimers with VEGF 165, with pro-angiogenic activity, and their over-expression has been described in the conditioned media of some tumor cell lines (Cao Y et al. J Biol Chem 271:3154, 1996), and associated to the evolution of rheumatoid arthritis and to primary inflammatory arthropathies, in general (Bottomley M J et al. Clin Exp Immunol 119:182, 2000).
- The over-expression of the rest of the members of the VEGF family, less studied, is also associated to a number of pathologies. VEGF-B has been related to breast, ovary, and kidney tumors, and to melanomas and fibrosarcomas (Sowter H M, et al. Lab. Invest. 77:607, 1997; Salven P Am. J. Pathol. 153:103, 1998, Gunningham S P et al. Cancer Res 61:3206, 2001). The differential expression of the VEGF-B 167 isoform in vitro has been reported in tumor cells of diverse origin (Li X, et al. Growth Factors 19:49, 2001). VEGF-C and VEGF-D are involved in the regulation of lymphatic vessels formation (Joukov V. et al EMBO J. 15: 290, 1996), and VEGF-C over-expression is associated to tissular edemas, to tumors of the breast, lung, head and neck, esophagus, and stomach, lymphomas, prostate cancer, and metastatic nodes (Kajita T, et al. Br J Cancer 85:255, 2001; Kitadi Y, et al Int J Cancer 93:662, 2001; Hashimoto I, et al. Br J Cancer 85:93, 2001; Kinoshita J, et al. Breast Cancer Res Treat 66:159, 2001; Ueda M, et al. Gynecol Oncol 82:162, 2001; Salven P Am. J. Pathol. 153:103, 1998; O-Charoenrat P et al. Cancer 92:556, 2001). In the case of VEGF-D, its over-expression by tumor cells is related to an in vivo increase of lymphatic vasculature in the tumors and the increase of metastasis in lymphatic nodes (Stacker S A, et al. Nat Med 7:186, 2001; Marconcini L et al. Proc Natl Acad Sci USA 96:9671, 1999).
- The alterations on endothelial cell function induced by the molecules of the VEGF family are mediated by their binding to cell receptors of the type tyrosine kinase class 3, that so far include: VEGFR1 (Flt1), VEGFR2 (KDR/Flk1), and VEGFR3 (Flt4) (Kaipainen A J. Exp. Med. 178:2077, 1993). The N-terminal domain 2 has been identified as responsible of the binding to the ligands, favoring the phosphorilation of the cytoplasmatic domain and transduction of the signal (Davis-Smyth T et al EMBO 15:4919, 1996).
- Ligands identified for VEGFR1 include VEGF-A, PIGF, and VEGF-B, in decreasing order of affinity (Shibuya M Int J Biochem Cell Biol 33: 409, 2001). In endothelial cells, this receptor captures the circulating VEGF (Gille H et al EMBO J. 19:4064, 2000). The binding of VEGF-A to the VEGFR1 expressed in cells of the hematopoyetic lineage affects significantly the activation of transcriptional factor NFκB in the precursors of dendritic cells, and in B and T lymphocytes. This last interaction is relevant in the in vivo establishment of an unfavorable immunologic balance, where dendritic cells maturation and the fraction of T lymphocytes are reduced, a phenomenon observed on immunosuppressed patients and in particular, with cancer (Dikov M M et al Canc Res 61:2015, 2001; Gabrilovich D et al. Blood 92:4150, 1998). Over-expression of VEGFR1 has been related with psoriasis, endometrial cancer and hepatocellular carcinoma (Detmar M, et al. J Exp Med 180:1141, 1994; Ng IO Am J Clin Patol 116:838, 2001; Yokoyama Y et al Gynecol Oncol 77:413, 2000).
- The VEGFR2 receptor (KDR/Flk1) mediates the biological effects of VEGF-A, and also binds VEGF-C and VEGF-D. This receptor is expressed differentially on activated endothelium and in some cell lines of tumor origin where it establishes autocrine pathways with the secreted VEGF. Apart from being involved in the already mentioned pathologies that are related with the over-expression of its ligands, the over-expression of VEGFR2 has been related with the progression of endometrial cancer (Giatromanolaki A et al, Cancer 92:2569, 2001), malignant mesothelioma (Strizzi L et al. J Pathol 193:468, 2001), astrocytic neoplasms (Carroll R S et al. Cancer 86:1335, 1999), primary breast cancer (Kranz A et al. Int J Cancer 84:293, 1999), intestinal type gastric cancer (Takahashi Y et al Clin Cancer Res 2:1679, 1996), multiform glioblastoma, anaplastic oligodendroglioma, and necrotic ependimoma (Chan A S et al. Am J Surg Pathol 22:816, 1998). Over-expression of KDR has also been associated to the autosomic disease VHL and to hemangioblastoma (Wizigmann-Voos S et al Cancer Res 55:1358, 1995), to the progression of diabetic retinopathy (Ishibashi T. Jpn J Opthalmol 44:323. 2000) and, in combination with Flt-1 over-expression, to the delayed-type hypersensitivity reactions (Brown L F et al J Immunol 154:2801, 1995).
- Lymphangiogenesis mediated by VEGF-C and VEGF-D results from their binding to the FLT4 receptor or VEGFR3, expressed in the lymphatic endothelium. In some cases, even when over-expression of the ligands is not present, the over-expression of the receptor has been related to an adverse prognosis in the course of a group of pathologic entities, including: diabetic retinopathy (Smith G. Br J Opthalmol 1999 April; 83(4):486-94), chronic inflammation and ulcers (Paavonen K et al, Am J Pathol 156:1499, 2000), the establishment of metastasis in lymphatic nodes and progression of breast cancer (Gunningham S P. Clin Cancer Res 6:4278, 2000 Valtola R et al. Am J Pathol 154:1381, 1999), associated to nasopharyngeal tumors and squamous oral carcinomas (Saaristo A et al. Am J Pathol 157:7, 2000; Moriyama M et al. Oral Oncol 33:369, 1997). Moreover, the over-expression of VEGFR3 is a sensitive marker of Kaposi sarcoma, type Dabska hemangioendothelioma and of cutaneous lymphangiomatosis (Folpe A L et al. Mod Pathol 13:180, 2000; Lymboussaki A et al. Am J Pathol 153:395, 1998).
- Recently, two receptors where identified for VEGF named NRP1 and NRP2. These belong to the neurophilins family (NRP), and act as co-receptors for some specific isoforms of proteins of the VEGF family: VEGF-A145 VEGF-A165, VEGF-B167 and PIGF1, increasing their mitogenic capacity. The expression of NRP1 has become a marker of the aggressiveness of prostate cancer, has been related to the increase of angiogenesis in melanomas, and with apoptosis escape events in breast cancer (Latil A et al. Int J Cancer 89:167, 2000; Lacal P M J Invest Dermatol 115:1000, 2000; Bachelder RE Cancer Res 61:5736, 2001). The coordinate over-expression of NRP1, KDR, and VEGF-A165 have been related to the fibrovascular proliferation in diabetic retinopathy cases and rheumatoid arthritis (Ishida S. et al. Invest Opthalmol Vis Sci 41: 1649, 2000; Ikeda M. Et al. J Pathol 191:426, 2000). NRP2 is over-expressed in osteosarcomas where it promotes angiogenesis and tumor growth (Handa A et al. Int J Oncol 17:291, 2000).
- Most of the therapeutic strategies based on angiogenesis inhibition, especially in cancer treatment, are based in the blockade of molecules of the VEGF family and their receptors, with clinical trials in course using: (1) monoclonal antibodies blocking VEGF or the KDR receptor, (2) metalloproteinase inhibitors, as Neovastat and Prinomastat, (3) VEGF inhibitors as Thalidomide, Suramin, Troponin I, and IFN-α and Neovastat, (4) blockers of VEGF receptors as SU5416, FTK787 and SU6668, (5) inducers of tumor endothelium apoptosis, as Endostatin and CA4-P, and (6) ribozymes that decrease VEGF or VEGF receptors expression (Angiozyme). Due to the high homology between human VEGF and its receptors KDR and Flt-1 with their murine homologs (˜90%, 81%, and 89%, respectively), many animal models are used routinely to evaluate the preclinical effectiveness of antiangiogenic compounds directed to this system (Hicklin D J et al. DDT 6:517, 2001).
- Passive administration of antibodies to VEGF or VEGFRs is successfully tested in different clinical phases in humans (Hicklin D J et al. DDT 6:517, 2001). The anti-VEGF humanized monoclonal antibody A.4.6.1 (Genentech, San Francisco, United States) is in phase III clinical trial for the treatment of colon, breast, kidney, and lung tumors (Kim, K J et al. Nature 362:841, 1993; Boersig C. R&D Directions October 7:44, 2001). In particular, for the case of the KDR receptor, a monoclonal antibody has been developed (IMC-1C11, ImClone) that recognizes the N-terminal extracellular domain of this receptor, and inhibits proliferation and migration of leukemic human cells, increasing survival of xenotransplanted mice. At present, its effect is being studied in patients with colon cancer metastasis (Dias S et al. J Clin Invest 106:511, 2000). In the aforementioned trials, the absence of concomitant adverse effects with the application of these monoclonal antibodies has been demonstrated.
- Notwithstanding the previous, a therapeutic modality not yet employed for the blockade of neoangionegesis is specific active immunotherapy (SAI). In the SAI of cancer, antigens as peptides, proteins or DNA are employed, mixed with appropriate adjuvants. SAI procedures pursue the stimulation of an immune response, both of the humoral (activation of B-lymphocytes), and cellular types (activation of T helper, and cytotoxic lymphocytes, and natural killer cells), associated to dendritic cell function as professional presenting cells in the MCHI and MHC II contexts (Bystryn J C, Medscape Hematology-Oncology 4:1, 2001; Parker, K C et al., J. Immunol 152:163, 1994; Nestle F O et al., Nature Medicine 7:761, 2001; Timmerman J M, Annual Review Medicine 50:507, 1999).
- SAI is a rapidly growing field of experimental and clinical research, with attractive applications, especially in oncology, where more than 60 undergoing clinical trials based in procedures of SAI are reported, which surpass at present the clinical trials based on the use of monoclonal antibodies. In the particular case of cancer, the antigens used as immunogens for SAI are selected because of their physiological relevance and difficulty of being substituted in the processes of tumor phenotypic drift (Bodey B et al., Anticancer Research 20: 2665, 2000), and because of their high specific association with the growth and evolution of tumor tissues.
- The strategy of treating cancer using SAI also considers preferably the identification of antigens expressed in different tumor types, what could increase the number of indications for the same vaccine preparation. Examples of these are carcinoembryonic antigen (CEA), HER2-neu, human telomerase, and gangliosides (Greener M., Mol Med Today 6:257 2000; Rice J, et al. J Immunol 167:1558, 2001; Carr A et al, Melanoma Res 11:219, 2001; Murray J L, et al. Semin Oncol 27:71, 2000).
- In human tumors, VEGF is over-express in the tumor compartment (Ferrara, N. Curr. Top. Microbiol. Immunol. 237:1, 1999), and high levels of VEGF and its receptors have been demonstrated in the tumor-associated vasculature (Brekken R A. J Control Release 74:173, 2001). The stromal cells also produce VEGF in response to the stimulus of transformed cells, with the result that when tumor cells are removed, VEGF levels persist in the patients. The presence of VEGF and its receptors have a practical value for the establishment of prognosis and staging in cases of prostate, cervix, and breast tumors (George D J et al. Clin Cancer Res 7:1932, 2001; Dobbs S P et al. Br J Cancer 76:1410, 1997; Callagy G et al. Appl Immunohistochem Mol Morphol 8:104, 2000). On the other hand, VEGF is also within the group of soluble factors that, together with other cytokines like IL-10, TNF-α and TGF-β, (Ohm J E y Carbone D P, Immunol Res 23:263, 2001), could be implicated in the immunosuppression that characterizes cancer patients (Staveley K, et al. Proc Natl Acad Sci USA 95:1178, 1998; Lee K H, et al. J Immunol 161:4183, 1998). This immunosuppressive effect seems to be related to its binding to the Flt1 receptor (Gabrilovich D et al. Blood 92:4150, 1998).
- The present invention describes procedures of SAI in experimental tumors using molecules of the VEGF family and their receptors. The antitumoral effects obtained could be based in at least four different mechanisms, without discarding their possible combinations: (a) direct destruction of cancer and stromal cells producing VEGF, by cytotoxic lymphocytes, (b) damaging of endothelial cells of tumor-associated vessels due to the capture or neutralization of the circulating VEGF via antibodies, (c) direct destruction of endothelial cells that express VEGF receptors, by cytotoxic lymphocytes or complement fixing antibodies, (d) activation of a local immune response as a consequence of the capture or neutralization of circulating VEGF, and its consequent elimination of its immunosuppressive effects.
- Ideally, these treatments could be used to diminish or avoid the appearance of metastasis, to reduce or eliminate primary tumors as a first or second line therapy, in combination or not with other anti-tumor agents.
- Active immunization directed to VEGF family and their receptors could also be efficient in the single or combined therapy of acute and chronic inflammatory processes (asthma, respiratory distress, endometriosis, atherosclerosis, tissular edema), infectious diseases (Hepatitis, Kaposi sarcoma), autoimmune diseases (diabetes, psoriasis, rheumatoid arthritis, thyroiditis, synovitis), diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma, among others.
- According to the present invention, the in vivo administration of oligonucleotide sequences encoding for proteins of the VEGF family, their receptors, co-receptors or their fragments, as well as of their polypeptidic variants, induces a cellular and humoral immune response with antiangiogenic and antitumoral effect.
- Immunogens of polypeptidic nature of interest for the present invention, as well as their fragments, can be isolated from their natural sources or obtained by synthesis or recombinant DNA technology. These polypeptides can also be produced fused to proteins with acknowledged adjuvant activity like p64K (R. Silva et al U.S. Pat. No. 5,286,484 y EP 0474313), or can be covalently bound to them after their individual obtainment. Other available strategy in these cases is the obtainment of the natural polypeptide, its mutated or modified variants, and their fragments, as a part of loops exposed or not in bacterial proteins like OMP1, which are part of immunostimulatory preparations, in this particular case VSSP (R. Perez et al U.S. Pat. No. 5,788,985 y 6149921). Furthermore, it is possible to obtain the polypeptidic immunogen exposed in the surface of a viral particle (HbsAg, VP2 of parvovirus, etc.), bound to specific peptides that target cells or organs specialized in the induction of the immune response (CTLA4, Fc segment of the Ig, etc.), or to proteins capable of increasing biodistribution like VP22.
- The principal natural sources of the proteins of interest for the present invention are predominantly expressed in placenta, activated endothelial cells, and tumor cells. The mRNA of these cells or tissues is used to obtain complementary DNA (cDNA) by known methods. The extracted RNA is used as template for the amplification through the polymerase chain reaction (PCR) of the cDNA corresponding to the selected antigen. In each case, primers used are designed according to the characteristics of the vector where the cDNA is going to be inserted and to the previously reported sequences of the protein of interest. Alternatively, and preferably in the case of the receptors amplified by PCR, that are the largest size antigens that are used in the present invention, the coding regions are amplified in two or more overlapping fragments. These fragments include a common ligation site used to assemble the intact DNA, starting with its fragments.
- An alternative for the cloning of the antigens of interest is the selection from commercially available DNA libraries derived from human endothelium, or from tumors of this same origin. In some cases, it might be desirable to mutate some of the antigens object of the present invention, in order to avoid, especially with the VEGF family, an angiogenesis induction event produced by vaccination. These mutations are made preferably in the receptor binding sites already described in the literature. For this, primers are designed that cover both ends of the desired molecule, and the PCR products are used as template to obtain the mutated molecule. These mutated variants lack biological activity but reproduce the immunogenic properties of the selected antigen.
- The cDNA molecules obtained by the methods described earlier are administered in a proper vector, being this a virus, a plasmid, a bacterial artificial chromosome, or similar. The vector carries the elements needed for the adequate expression of the gene in target cells, as well as the rest of elements that allows it to be produced in the host cellular system according to its nature. DNA molecules of the present invention might contain one or more genes of interest, constituted by one or more nucleic acids (cDNA, gDNA, synthetic or semi-synthetic DNA, or similar) that when transcribed or translated (when appropriate) in target cells generates the products with therapeutic/vaccine value.
- Generally, the gene of the vaccine therapeutic product according to the invention is under the control of a transcriptional promoter that is functional in the target cell or the organism (mammals), as well as of a 3′ end region that contains the signals needed for termination and polyadenilation of the mRNA of the product of interest, allowing its expression. The promoter can be the natural promoter of the gene or a heterologous promoter transcriptionally active in the target cell. The promoter can be from eukaryotic or viral origin. Among eukaryotic promoters, it is possible to use any promoter or derived sequence that stimulates or represses the gene transcription, specifically or not, inducible or not, in a strong or weak manner. Additionally, the promoter region can be modified by the insertion of activators or inductor sequences, allowing the tissue-specific or predominant expression of the gene in question.
- Besides, the gene of interest can contain a signal sequence for subcellular localization, in a way that its cellular localization or secretion could be modified in the cell where it is expressed, or elsewhere, once synthesized. It can also contain a sequence encoding for a region of specific binding to a ligand specific of immune tissue, being directed to the site where the response is generated, with the obtainment of the therapeutic/vaccine effect.
- Additionally, the gene of interest can be preceded by the coding sequence for the mRNA replication machinery, in a way that mRNA is amplified in the target cell, increasing the expression of said gene, and with it, of the therapeutic/vaccine effect according to the invention. The replication machinery in question could of alphavirus origin (Schlesinger S., Expert Opin Biol Ther. 1:177, 2001), more specifically derived from the Sindbis or Semliki viruses, or similar. In this particular case, the gene of interest is under the transcriptional control of a subgenomic promoter that allows the amplification of its mRNA in target cells, once the molecules according to the present invention have been internalized. Besides, the DNA vector might contain sequences that permit the replication of the molecules object of the present invention in mammalian cells. This allows an increase in the expression levels and/or of the therapeutic/vaccine effect (Collings A., Vaccine 18: 4601, 1999)
- The DNA vector can be purified using standard techniques for plasmid DNA purification. These techniques include the method of purification by cesium chloride density gradient, in the presence of ethidium bromide, or alternatively, the use of ionic exchange columns or any other exchanger or method to separate DNA molecules (Ferreira G N, et al, Trends Biotechnol. 18:380, 2000).
- The present invention includes the use of plasmidic DNA vectors, preferably those of the PAEC family of compact vectors for DNA immunization and gene therapy in humans (Herrera et al, Biochem. Biophys. Res. Commu. 279: 548, 2000). This family comprises vectors pAEC-K6 (Access number AJ278712), pAEC-M7 (Access number AJ278713), pAEC-Δ2 (Access number AJ278714), pAEC-SPE (Access number AJ278715) and pAEC-SPT (Access number AJ278716). These vectors contain only the essential elements for the expression of the product of interest in mammalian cells, including human cells, and a replication unit in Escherichia coli. The transcriptional unit is formed by the immediate early promoter of human cytomegalovirus (CMV), a versatile multicloning site for the insertion of the product of interest, and the sequences for transcriptional termination and polyadenilation derived from simian virus 40 (SV40). In the replication unit, the vector contains the gene for kanamycin resistance (Tn903), and a pUC19 replication origin (ColE1), in order to guarantee a high copy number and the selection of the bacteria that bear the plasmid of interest.
- Furthermore, the present invention includes the use of plasmidic DNA vectors, preferably those of the PMAE family of compact vectors for DNA immunization in humans. These contain the same functional elements in bacteria as PAEC series, as well as the CMV immediate early promoter and the multicloning site. Additionally, they bear a synthetic intron and a synthetic sequence for transcription termination and polyadenilation, derived from rabbit β-globin. It has been reported that with sequences similar to the latter it is possible to obtain higher expression levels of the cloned gene (Norman J A et al, Vaccine 15: 801, 1997). Moreover, the vectors of this series include consecutive repetitions of immunostimulatory sequences (CpG motives), that stimulate innate immune system in both mice and humans, with the consequent activation of a humoral and cellular response against the molecule of interest (Krieg A M, Vaccine 19:618, 2001).
- The immunization with recombinant virus (adenovirus, adeno-associated, vaccinia, chickenpox virus, canarypox virus, among others) produces a potent cytotoxic cellular response in the hosts. To introduce the sequence of interest in the recombinant virus vectors that have integration sequences and promoters that are particular for each virus type, are used. This strategy is also included in the scope of the present invention, and chickenpox virus and the pFP67xgpt vector are preferably used. The pFP67xgpt vector is used to clone genes under a strong early/late promoter of synthetic nature between the open reading frames 6 and 7 of a fragment of 11.2 kB BamHI of the chickenpox virus FP9. This plasmid also contains the Ecogpt controlled by the vaccinia promoter p7.5K, which is used to identify recombinant virus.
- Other alternative of the present invention consists of the immunization with proteins of the VEGF family and their receptors and/or co-receptors. cDNA molecules obtained as previously described are cloned in vectors for expression in virus, yeast, phage, plants, or superior cells, in order to obtain the protein variants of the antigens, after their sequence has been verified by the traditional methods of automatic sequencing. Several vectors for expression have been described and used for the obtainment of recombinant proteins. These vectors contain, at least, a sequence that controls the expression operatively linked to the sequence of the DNA or fragment to be expressed. Examples of sequences useful for the control of expression are: the systems lac, trp, tac, and trc, the promoter regions and the principal operator of lambda phage, the controller region of the surface protein fd, the glycolytic promoters of yeast (for example, the 3-phosphoglicerate kinase), the promoters of yeast acid phosphatase (for example, Pho5), the yeast promoters for the mating alpha factor, and the promoters derived from polyoma, adenovirus, retrovirus, simian virus (for example, the early/late promoters of SV40), and other known sequences that regulate the expression of genes in prokaryotic and eukaryotic cells, their viruses, and their combinations.
- The hosts used for the replication of these vectors and the obtainment of the recombinant proteins object of the present invention include prokaryotic and eukaryotic cells. The prokaryotic comprise E. coli (DHI, MRCI, HB101, W3110, SG-936, X1776, X2282, DH5a), Pseudomonas, Bacillus subtilis, Streptomices, and others. The eukaryotic cells include yeast and fungi, insects, animal cells (for example, COS-7 and CHO), human, and plant cells, and tissue cultures, among others. After the expression in the system of choice in an adequate media, the polypeptides or peptides can be isolated by known procedures.
- Even when vaccination with naked DNA or proteins has shown to be effective in certain animal models, the patients affected by tumors or autoimmune diseases present a challenge to the therapeutic strategy proposed by the present invention. To favor the immune response, the DNA or protein vaccines can be combined with immunopotentiators already described like: mineral salts (ex., Aluminum hydroxide, aluminum phosphate, calcium phosphate); immunostimulators like: cytokines (ex., IL-2, IL-12, GM-CSF, IFN-α, IFN-γ, IL-18), molecules (ex., CD40, CD154, invariant chain of MHC type I, LFA3); saponins (ex., QS21), MDP derivatives, CpG oligos, LPS, MPL and polyphosphazenes; lipidic particles: like: emulsions (ex., Freund, SAF, MF59), liposomes, virosomes, iscoms, co-chelators; microparticular adjuvants like PLG microparticles, poloxamers, of viral type (ex., HBcAg, HCcAg, HBsAg), and of bacterial type (ie., VSSP, OPC); and mucosal adjuvants like heat-labile enterotoxin (LT), cholera toxin, and mutant toxins (ex., LTK63 y LTR72), microparticles and polymerized liposomes. In the case of DNA vaccination, the expression of the antigen of interest could be combined with some of the immunopotentiator molecules already mentioned, on a bi-cistronic vector.
- The experimental situations detailed in the examples demonstrate that DNA can be coupled in a non-covalent manner to some of the mentioned particles and that the use of these mixtures reduce the optimal concentration to obtain an anti-tumor response, similar to those described for higher doses of naked DNA.
- For the therapeutic applications, the vaccine preparations of the present invention are administered to a mammal, preferably a human, in a dose pharmaceutically acceptable, by the following routes: mucosal, subcutaneous, intramuscular, peritoneal, intra-lymphatic, topic, and by inhalation, among others. These could be administered on the tissue interstitial space, including: muscle, skin, brain, lung, liver, bone marrow, spleen, thymus, heart, lymph nodes, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testicles, ovary, uterus, rectum, eye, glands, and connective tissue. In the case of vectors for oligonucleotide transfer, their expression is preferably directed to somatic differentiated cells, though they may be directed to non-differentiated or less differentiated cells like skin fibroblasts and blood pluripotent cells.
- The doses of the immunogen could be administered in pharmaceutically accepted vehicles without toxicity or therapeutic effects. Examples of these vehicles include: ionic exchangers, alumina, aluminum esthearates, lecitine, seric proteins like albumin, buffer solutions, like phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated fatty acids of plant origin, water, salts, or electrolites, like protamine sulphate, di-sodic hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pirrolidone, substances base on cellulose and polyethylene glycol. In the present invention, preferably phosphate buffers as vehicles of the vaccine preparations are used.
- In the case of the use of proteins and peptides, these can be conjugated in covalent or non-covalent manner to molecules known as carriers that act like adjuvants. Among these molecules are: KLH, p64K, OPC (Musacchio A et al, Vaccine 19; 3692, 2001), and VSSP. The combination of naked DNA, viral vectors, and protein immunogens is an alternative also included within the scope of the present invention. In an advantageous manner, plasmid DNA administration allows the generation of formulations with one or more molecules of interest in the vaccine preparation. Thus, molecules according to the present invention can be administered in vaccine schedules through the combination of different types of vectors (variant of induction re-stimulation, with DNA, proteins, viral vectors)
- DNA vectors could be directly administered to the patient, or host cells can be in vivo or ex vivo modified with these vectors. This last strategy can be combined with the insertion by site-specific recombination or the immunization by somatic transgenesis that directs the vector expression to specific cells. Furthermore, bacterial hosts of DNA vectors could be used as their vehicles of transfer in vivo.
- In this way, the molecules carrying the genes according to the present invention could be used in the form of naked DNA, or in combination with different vectors: chemical/biochemical/biologic, natural/synthetic or recombinant. These molecules can be coupled or combined with cationic peptides, compacting molecules (ex. PEG, PEI), nuclear localization peptides (NLP), etc. These could be administered also together with cations capable of forming DNA precipitates, as a part of liposomal preparations to which the molecules have been added previously to the membrane fusion, and in synthetic vectors of lipid nature, or formed by cationic polymers (ex. DOGS or DOTMA). For the administration of the DNA vectors, chimeric proteins able to compact DNA and mediate the transport of the complex formed, and its selective endocytosis by specific cells, can also be used. DNA molecules carrying the therapeutic/vaccine genes according to the invention could be used for the genetic transfer to cells using physical methods of transfer, like particles bombardment, electroporation (in vitro, in vivo or ex vivo), or directly in vivo by topic application, inhalation by particulation, etc. The live vectors include adenoviral particles or the same hosts where the molecules according to the present invention have been generated.
- The doses of polypeptides and/or oligonucleotides to be used can be established according to different parameters, in particular depending on the gene or protein administered as an immunogen, the route of administration, the pathology to be treated, the period of treatment, and in the case of using oligonucleotides, of the vector used for immunization. A change in dose schedule or administration route different to those described in the following examples, do not separate from the principle or precept of the present invention, being possible to achieve an optimization of the immunization schemes to obtain a better response.
- The present invention has advantages over passive immunotherapy, which is in advanced phases of clinical trials using the same molecules as targets. In comparison with passive transfer of immunity through the administration of monoclonal antibodies (ex. Anti-VEGF), the immunization, be it with the protein or the oligonucleotide, has the advantage of inducing the endogen production of antibodies and in addition the proliferation and expansion of specific cytotoxic CD8+ lymphocytes.
- The present invention has advantages over the therapeutic strategies directed to block VEGF-VEGFRs system, mainly because these strategies only diminish the levels of circulating VEGF or block KDR. The strategy proposed, apart from achieving the mentioned effects, also destroys the source of VEGF (that is, the tumor cells and associated stroma) and/or the cells expressing their receptors (tumor endothelium and some tumor cells). Previous work done in this area only describe a humoral response as a principal component of the observed effect. Without the intention of limiting the scope of the present invention to a particular mechanism, the examples show that, besides from the humoral specific response, the vaccine compositions are able to elicit a CD8+ cellular response that cooperates with the humoral response; and that in the tumor context, the combination of both are relevant to obtain an ant-tumor effect, the previous observed in example 9.
- It is possible that the cytotoxic cellular response is mediated by the recognition of some of the peptides that appear in Tables 1 and 2. In these, some peptidic segments appear, that could be relevant in the cellular response directed to selected targets in VEGF family, its receptors and co-receptors. This information was obtained through computer analyses on public databases from NIH and Heidelberg Institute (http://bimas.dcrt.nih.gov/molbio/hla_bind, and www.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm) using BIMAS and SYFPHEITI software, respectively. The peptides marked and other sequences derived from the antigens of interest could be used for the active immunotherapy of the already described pathologies, as a single or combined treatment, and as part or not of molecules with adjuvant capacities. These peptides can also be used in their oligonucleotide variants with vaccine purposes.
- The methods to inhibit angiogenesis and the pathologic conditions associated to this event, comprise the administration of an effective amount of the DNA or protein of some of the molecules described in this invention, by any of the routes, and with the use of some of the previously described immunopotentiators or adjuvants, to a mammal. This mammal is preferably a human.
- A non-reversible and unregulated increase of angiogenesis has been related to a wide group of diseases. The system that comprises the VEGF family, its receptors and co-receptors is over-expressed in many of these pathological conditions, as it has been described before. In this way, the therapeutic strategies proposed by the present invention result effective in the treatment of: (a) cancer (both primary tumors and their metastasis), (b) acute and chronic inflammatory processes like asthma, respiratory distress, endometriosis, atherosclerosis, and tissular edema, (c) diseases of infectious origin like Hepatitis and Kaposi sarcoma, (d) autoimmune diseases like diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and (e) other diseases and states such as diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma and angiofibroma.
- Particularly in the case of cancer, vaccination with the immunogens proposed by the present invention results effective in the treatment of carcinomas, sarcomas and vascularized tumors. Some examples of tumors that can be treated with the proposed strategies include epidermoid tumors, squamous tumors like those of the head and neck, and colorectal, prostate, breast, lung (including small and non-small cells), pancreas, thyroid, ovary, and liver tumors. These methods are also effective in the treatment of other types of tumors, like Kaposi sarcoma, central nervous system neoplasia (neuroblastoma, capillary hemangioma, meningioma and brain metastasis), melanomas, renal and gastrointestinal carcinomas, rhabdomyosarcoma, glioblastoma and leiomiosarcoma.
- Specifically the use of VEGF-A and/or their receptors VEGFR-1 and VEGFR-2 as immunogen is useful for the treatment of: tumors of different origins and localizations and their metastasis, of hemangioma, of endometriosis, of tissue edemas, of chronic inflammatory processes like ulcerative colitis and Crohn's disease, of, atherosclerosis, of rheumatoid arthritis and osteoarthritis, of inflammatory arthropathies, psoriasis, respiratory distress, asthma, thyroidits, of diabetic and newborn retinopathies, macular degeneration, and glaucoma, of the autosomic VHL disease, of obesity, and of the rejection of some organ transplants. On the other hand, a response vs PIGF is useful in cases of rheumatoid arthritis and in general for the treatment of primary inflammatory arthropathies.
- In the case of VEGF-B, its use as immunogen results useful in cases of breast, ovary, and kidney tumors, and for melanoma and fibrosarcoma. The use of VEGF-C and its receptor VEGFR-3 results useful in the treatment of tissular edema, diabetic retinopathy, chronic inflammation, ulcers, and tumors of the breast, lung, head and neck, esophagus, stomach, lymphomas, and prostate, metastatic nodules and Kaposi sarcoma, Dabska type hemangioendothelioma and of the cutaneous lymphangiomatosis. Immunization with VEGF-D can be used specifically for the treatment of lymphatic node metastasis.
- The use of NRP1 and NRP2 co-receptors in mammal immunization results useful for the treatment, in particular, of fibrovascular proliferation in prostate cancer, melanoma, osteosarcoma, breast cancer metastasis, diabetic retinopathy, and rheumatoid arthritis.
- The studies based on the passive immunotherapy by administration of antibodies have shown that the combination of antibodies vs VEGF-A and KDR is more effective in models of syngeneic tumors. Thus, the use of two or more of the immunogens proposed in the present invention provides an especially efficient treatment for the inhibition of angiogenesis and tumor growth. These immunogens can be administered in an individual manner or by pairs using bi-cistronic vectors by the already mentioned pathways. Furthermore, vaccine compositions of the invention can be used together with, or in sequential manner, with drugs or chemotherapeutic agents, that offer a benefit to the condition under treatment.
- The results described below demonstrate that the anti-angiogenic and anti-tumor responses are mediated by a cooperation of the humoral and cellular responses. In particular, VEGF and its receptor are involved in the process of maturation of dendritic cells and act on B and T lymphocytes precursors. Example 10 demonstrates that the proposed therapeutic strategy, apart from diminishing the levels of VEGF in sera also contributes to the normalization of the proportions of B and T lymphocytes, and of mature dendritic cells. This effect favors the presentation of tumor antigens within the MHC I context, improving the quality and intensity of the immune anti-tumor response directed not only to the immunogen, but also to the other tumor-associated, tumor-specific, and over-expressed antigens, in the tumor context.
-
TABLE 1 Estimation of the VEGF protein family MHCI associated peptides in the context of HLAA.0201 A.-Using BIMAS software VEGF-A VEGF-B VEGF-C VEGF-D PIGF Sequence Kd Sequence Kd Sequence Kd Sequence Kd Sequence Kd LLSWVHWSL 272 LLLAALLQL 309 YLSKTLFEI 640 FMMLYVQLV 1966 RLFPCFLQL 150 ALLLYLHHA 42 QLAPAQAPV 70 TLFEITVPL 324 KLWRCRLRL 620 VVSEYPSEV 42 WSLALLLYL 30 QLVPSCVTV 70 VLYPEYWKM 304 QLFEISVPL 324 VMRLFPCFL 42 FLQHNKCEC 23 LMGTVAKQL 26 CMNTSTSYL 85 YISKQLFEI 88 RALERLVDV 34 WVHWSLALL 20 LLAALLQLA 19 KLFPSQCGA 64 CMNTSTSYI 41 VELTFSQHV 32 FLLSWVHWS 16 LLQLAPAQA 8 LLGFFSVAC 32 VLQEENPLA 35 AVPPQQWAL 14 RQLELNERT 6 WSWIDVYT 6 SLPATLPQC 11 WVVVNVFMM 27 LQLLAGLAL 14 NITMQIMRI 3 CVPTGQHQV 6 GLQCMNTST 7 VNVFMMLYV 10 RSGDRPSYV 10 YCHPIETLV 2 KQLVPSCVT 4 AAFESGLDL 4 SLICMNTST 7 LLAGLALPA 8 IEYIFKPSC 2 VVVPLTVEL 3 EQLRSVSSV 4 CVLQEENPL 7 CVPVETANV 6 B.-Using SYFPEITHI software VEGF-A VEGF-B VEGF-C VEGF-D PIGF Sequence Score Sequence Score Sequence Score Sequence Score Sequence Score LLSWVHWSL 24 LLLAALLQL 29 TLFEITVPL 27 FMMLYVQLV 25 ALERLVDVV 26 ALLLYLHHA 24 QLAPAQAPV 26 DLEEQLRSV 26 QLFEISVPL 25 RLFPCFLQL 24 WVHWSLALL 20 QLVPSCVTV 26 YLSKTLFEI 26 YISKQLFEI 24 RALERLVDV 24 SLALLLYLH 20 VVVPLTVEL 24 ALLPGPREA 24 KLWRCRLRL 23 LLAGLALPA 22 SYCHPIETL 19 LLRRLLLAA 23 CMNTSTSYL 21 RAASSLEEL 22 LAGLALPAV 22 NITMQIMRI 19 LLAALLQLA 23 DICGPNKEL 21 SLEELLRIT 22 VMRLFPCFL 20 FLLSWVHWS 18 FLRCQGRGL 22 AAAAFESGL 20 ATFYDIETL 22 CFLQLLAGL 20 WSLALLLYL 18 LTVELMGTV 21 AAFESGLDL 20 EISVPLTSV 22 QLLAGLALP 20 HPIETLVDI 18 LRRLLLAAL 20 VLYPEYWKM 20 SLICMNTST 20 SAGNGSSEV 20 CNDEGLECV 18 LMGTVAKQL 19 IIRRSLPAT 20 VPLTSVPEL 20 VVSEYPSEV 20 Note: Values in bold correspond to those peptides or their regions, which coincide in both predictions. -
TABLE 2 Estimation of VEGF family receptors MHCI associated peptides in the context of HLAA.0201 A.-Using BIMAS software VEGFR-1 VEGFR-2 VEGFR-3 NRP-1 NRP-2 Sequence Kd Sequence Kd Sequence Kd Sequence Kd Sequence Kd FLYRDVTWI 1942 VLLWEIFSL 1792 VLLWEIFSL 1793 GLLRFVTAV 2249 WMYDHAKWL 5121 VLLWEIFSL 1792 SLQDQGDYV 769 RLLEEKSGV 1055 VLLGAVCGV 1006 ILQFLIFDL 484 KLLRGHTLV 901 VLLAVALWL 739 VLWPDGQEV 981 WMPENIRLV 436 YLQVDLRFL 247 GLLTCEATV 257 AMFFWLLLV 427 NLTDLLVNV 656 GILSMVFYT 278 ALYFSRHQV 223 TLFWLLLTL 182 VIAMFFWLL 270 KQAERGKWV 557 LLCAVLALV 272 NMLGMLSGL 131 ILLSENNVV 179 ILLSEKNW 179 GVIAVFFWV 369 VLLHKSLKL 134 WLYTLDPIL 129 TLNLTIMNV 160 LLAVALWLC 146 KLVIQNANV 243 GMLGMVSGL 131 DIWDGIPHV 56 CVAATLFWL 137 KNLDTLWKL 128 ALWNSAAGL 177 FQLTGGTTV 120 KMEIILQFL 44 LLSIKQSNV 118 AVIAMFFWL 113 TLSLSIPRV 160 VLATEKPTV 118 VLNKLHAPL 36 SLQDSGTYA 112 LLLVIILRT 108 SQHDLGSYV 159 GPFLFIKFV 81 LLGATCAGL 36 B.-Using SYFPEITHI software VEGFR-1 VEGFR-2 VEGFR-3 NRP-1 NRP-2 Sequence Score Sequence Score Sequence Score Sequence Score Sequence Score TLFWLLLTL 29 VLLWEIFSL 29 VLLWEIFSL 29 VLLGAVCGV 30 NMLGMLSGL 27 VLLWEIFSL 29 LLVIILRTV 28 SIPGLNVTL 27 GLLRFVTAV 29 ILQFLIFDL 26 ILGPGSSTL 28 GLFCKTLTI 26 NLTDLLVNV 27 LLCAVLALV 28 DIWDGIPHV 26 LLCALLSCL 27 SIMYIVVVV 26 VLWPDGQEV 26 GMLGMVSGL 28 YLQVDLRFL 26 GLLTCEATV 27 IILVGTAVI 26 LLPRKSLEL 26 ALGVLLGAV 28 TLDPILITI 26 LLRGHTLVL 27 ALMSELKIL 26 ALWNSAAGL 26 VLLHKSLKL 27 ILAKPKMEI 25 ALMTELKIL 26 AASVGLPSV 25 IMDPGEVPL 26 VLATEKPTV 26 VLNKLHAPL 25 KLLRGHTLV 25 SISNLNVSL 25 RLWLCLGLL 25 QLTGGTTVL 25 LLGATCAGL 25 TLNLTIMNV 25 AMFFWLLLV 25 LIYFYVTTI 25 VLLGAVCGV 30 ALYFSRHQV 23 ILLSENNVV 25 ILLSEKNW 25 LLEGQPVLL 25 GLLRFVTAV 29 GIGMRLEVL 23 Note: Values in bold correspond to those peptides or regions, which coincide in both predictions. - VEGF isoforms were cloned applying the polymerase chain reaction (PCR) using as template a cDNA obtained from a previous isolation of mRNA of CaSki cell line (ATCC CRL 1550), according to the manufacturer instructions (Perkin-Elmer), and utilizing primers SEQ ID1 and SEQ ID2. Bands corresponding to the amplification products of VEGF isoforms 121, 165 and 189 were extracted from 2% agarose gels. After band digestion with endonucleases BamHI and EcoRI, the cDNAs from the VEGF isoforms were purified and cloned independently in the PAECΔ2 vector (proprietary vector of CIGB). Resulting plasmids were sequenced and determined to have no mutations with respect to the aminoacid sequences reported by the EMBL (www.embl-heidelberg.de) for the cloned isoforms. The cDNA corresponding to VEGF isoforms were subsequently cloned KpnI/EcoRV on the pMAE5Δ5 vector, that among other characteristics differs from pAECΔ2 by the presence of 5 immunostimulatory CpG sites.
- cDNA from a VEGF variant deficient for the binding to the KDR receptor (VEGFKDR(−)) was obtained by direct mutagenesis of the VEGF121 isoform previously cloned, as described by Siemeister G et al (Siemeister G et al. J Biol Chem 273:11115, 1998). The mutated variant was generated by PCR using the following primers:
- (A) Amplification of the 5′ terminal fragment (315 bp): using primers with sequences SEQ ID3 and SEQ ID4
- (B) Amplification of the 3′ terminal fragment (93 bp): using primers with sequences SEQ ID5 and SEQ ID6.
- The fragments thus amplified were purified as referred, and were used in equimolar concentrations as a template for a fusion PCR using the primers corresponding to sequences SEQ ID7 and SEQ ID8. Resultant cDNA containing the mutation was digested BamHI/EcoRI, and was purified, and cloned in pAECΔ2 vector. The mutations introduced were checked by sequencing, and the DNA corresponding to VEGFKDR(−) was subcloned KpnI/EcoRV in pMAE5Δ5 vector resulting in pMAE5Δ5 VEGFKDR(−).
- Plasmids used both in transfection and in animal vaccination were purified in endotoxin-free conditions, as described by Whalen R. et al. (Whalen R G y Davis H L. Clin Immunol Immunopathol 75:1, 1995). Briefly, DNA was purified using QIAGEN Endo-free systems following the manufacturer instructions, and the DNA was furthermore submitted to a second precipitation. Finally, DNA was dissolved in endotoxin-free PBS (SIGMA, USA) to a final concentration of 4 mg/mL.
- The cDNAs coding for the extracellular domain of KDR receptor of VEGF (KDR1-3) and for the transmembrane and intracellular domains of this receptor (KDR TC), were obtained from an RT-PCR using mRNA of the endothelial cell line HUVEC (Clonetic, USA), treated with human VEGF (Sigma) and Heparin (Sigma).
- In the case of the extracellular domains 1 to 3, the primers used correspond to sequences SEQ ID9 and SEQ ID10. After digestion of the amplified fragment (943 bp) with endonucleases BamHI and EcoRI, the cDNA coding for 1-3 domains of KDR was purified, and cloned in pAECΔ2 vector. Clones positive by restriction analysis were verified by sequencing of the corresponding DNA. The cDNA corresponding to KDR 1-3 was then subcloned KpnI/EcoRV in the already described pMAE5Δ5 vector (pMAE5Δ5 KDR1-3).
- For the cloning of transmembrane and cytosolic regions of the receptor a two-step strategy was designed. For the insertion of the first segment, the primers corresponding to SEQ ID11 and SEQ ID12 were used. After the XbaI/BgIII digestion of this 747 bp segment, the product was cloned in the pMAE5 vector, previously digested with the same enzymes, obtaining the plasmid PMAE5 KDR 747. This plasmid was digested BglII/NotI in order to insert the remaining carboxi-terminal fragment of 1091 bp that was amplified using the primers corresponding to sequences SEQ ID13 and SEQ ID14. Clones positive by restriction analysis were verified by DNA sequencing and denominated pMAE5 KDR C.
- 1.2.1 Cloning of the transmembrane and cytosolic regions of KDR in a viral vector For the cloning of transmembrane and cytosolic regions of VEGF receptor (KDR) on the chickenpox virus, the primers corresponding to sequences SEQ ID15 and SEQ ID16 were used. After digesting this 953 bp segment with StuI/SmaI enzymes, the product was cloned in the pFP67xgpt vector, previously digested with the same enzymes. In this same vector, digested SmaI/BamHI, the remaining 919 bp were inserted, that were amplified from the original cDNA using primers corresponding to sequences SEQ ID17 and SEQ ID18. Clones positive by restriction analysis were verified by DNA sequencing and denominated pFP67xgpt KDR C.
- Chickenpox virus (FWPVs) were replicated in chicken embryo fibroblasts (CEF), in DMEM medium supplemented with 2% of fetal bovine serum (FBS). The pFP67xgpt KDR C was transfected using Lipofectin (Gibco BRL, Grand Island, USA) in CEF previously infected with the attenuated strain FP9. After 24 hours, fresh medium was added and cells were cultured for other 3 to 4 days. After this time, cells were frozen-thawed three times. Recombinant viruses expressing the gene coding for the Ecogpt enzyme were purified in selective media with mycophenolic acid (25 μg/mL), xantine (250 μg/mL), and hypoxantine (15 μg/mL) (MXH). The correct inclusion of the gene in recombinant viruses was checked by PCR. Recombinant viruses were denominated FPKDRgpt and non-recombinants used as negative control FP.
- In order to confirm the potential of the constructions made to express the proteins in vivo, these were injected in the femoral quadriceps muscle of C57BL6 mice (3 per group)
- 1. pMAE5Δ5-VEGF121 (10 and 50 μg/mouse) in PBS pH 7.2
2. pMAE5Δ5-VEGF165 (10 and 50 μg/mouse) in PBS pH 7.2
3. pMAE5Δ5-VEGF189 (10 and 50 μg/mouse) in PBS pH 7.2
4. pMAE5Δ5-VEGFKDR(−) (10 and 50 μg/mouse) in PBS pH 7.2
5. pMAE5Δ5-KDR 1-3 (10 and 50 μg/mouse) in PBS pH 7.2
6. pMAE5 KDR C (10 and 50 μg/mouse) in PBS pH 7.2
7. FPKDRgpt (2.5*107 cfu) in PBS pH 7.2
8. PBS pH 7.2 (negative control) - 48 hours after injection the animals were sacrificed and injected muscles extracted in one piece. A part of the muscular tissue was homogenized in presence of protease inhibitors and non-ionic detergents. Presence of VEGF in protein extracts was analyzed by Dot-Blot and by Western-Blot using a polyclonal antibody that recognizes all human VEGF isoforms (sc-152G), following described procedures. RNA was extracted from the remaining muscular tissue using TRI-Reagent (SIGMA). A total of 20 μg of RNA from each experimental situation were submitted to electrophoresis in 1% agarose gels containing formaldehyde. RNA was transferred to a nylon filter (HYBOND) and hybridized with the cDNA of VEGF 121 isoform labeled with ATP32, that recognizes all VEGF isoforms, or with the cDNA of KDR similarly labeled. In both cases, filters were re-hybridized with the cDNA corresponding to a constitutive gene: the gliceraldehyde 3-phosphate dehydrogenase (GAPDH). In all the analyzed constructions bands corresponding to human VEGF and the cloned fragments of the KDR receptor were identified.
- Groups of 10 C57BL/6 mice were vaccinated or not with the following variants:
- 1. pMAE5Δ5-KDR 1-3 (1, 10, 50 and 100 μg/mouse) in PBS pH 7.2
2. pMAE5 KDR C (1, 10, 50 and 100 μg/mouse) in PBS pH 7.2
3. FPKDRgpt (2.5*107 cfu)
4. PBS pH 7.2 (negative control)
5. FP (2.5*107 cfu) (negative control group 3) - In every case, mice were immunized by intramuscular injection (im.) in the rear left foot with a total volume of 50 μl. All the animals were re-immunized 15 days later using the original immunization regime. The tumor challenge was developed thirty days after the last immunization, by a subcutaneous (sc.) injection of 104 cells of B16-F10 melanoma (ATCC, CRL-6475) in the right ventral zone of every animal. Tumor growth was monitored with three weekly measurements until animals started to die.
- In mice immunized with the pMAE5Δ5-KDR 1-3 plasmid a reduction of tumor size was evidenced at doses of 50 and 100 μg of DNA/mouse, significantly lower with respect to the negative control (Table 3). A survival analysis at day 33 revealed a significant increment (with respect to the negative control) of this parameter for the animals immunized with the said DNA doses of 50 and 100 μg per mouse, with respect to the un-immunized mice (group PBS pH7.2). In the case of pMAE5Δ5-KDR C (Table 3) a significant reduction of tumor volume was observed at the four doses used, with an increment in survival for doses from 100 to 10 μg/animal. The use of viral vectors reduced the volume and increased survival in the condition used for the FPKDRgpt construction (Table 3), in comparison to the respective negative control (group of mice immunized with the vector without insert FPgpt).
-
TABLE 3 Tumor volume and survival in mice immunized with the fragments of the VEGF receptor (KDR) gene. [DNA Tumor Vol. (mm3) Survival Group μg] Day 24 (Day 43) PMAE5Δ5- 100 424.0 ± 199.2 (***) (***) KDR1-3 50 756.32 ± 435.9 (***) (**) 10 1024.2 ± 397.1 (*) (ns) 1 1334.2 ± 620.7 (ns) (ns) pMAE5Δ5- 100 404.23 ± 200.0 (***) (***) KDR C 50 633.2 ± 365.2 (***) (***) 10 924.3 ± 437.1 (**) (*) 1 1114.2 ± 665.7 (*) (ns) FPKDRgpt 2.5*107 cfu 304.23 ± 152.0 (***) (***) FPgpt 2.5*107 cfu 1891.0 ± 726.0 (ns) (ns) PBS pH 7.2 — 1785.0 ± 826.0 — Note Tumor volume is reported as mean ± standard deviation (SD) of the measures performed on the animals of each group, statistical comparisons were performed using one-way ANOVA and a Bonferroni post-test. In the case of survival, the reported statistical significance was obtained using the log-rank test to compare each group with respect to the control group, in the indicated day. Statistical signification is indicated as (ns) p ≦ 0.05 non-significant; (*) p ≦ 0.05; (**) p ≦ 0.01; and (***) p ≦ 0.001. - Groups of 10 mice C57BL/6 were vaccinated or not with the following variants:
- 1. pAECΔ2-VEGF121 (1, 10, 50 and 100 μg/mouse) in PBS pH 7.2
2. pMAE5Δ5-VEGF121 (1, 10, 50 and 100 μg/mouse) in PBS pH 7.2
3. pMAE5Δ5-VEGF165 (1, 10, 50 and 100 μg/mouse) in PBS pH 7.2
4. pMAE5Δ5-VEGF189 (1, 10, 50 and 100 μg/mouse) in PBS pH 7.2
5. pMAE5Δ5 VEGFKDR(−) (1, 10, 50 and 100 μg/mouse) in PBS pH 7.2
6. PBS pH 7.2 (negative control) - In every case, mice were immunized by im. injection in the rear left foot with a total volume of 50 μl. All the animals were re-immunized 15 days later using the original immunization regime. The tumor challenge was developed thirty days after the last immunization, by a subcutaneous injection of 104 cells of B16-F10 melanoma (ATCC, CRL-6475) in the right ventral zone of every animal. Tumor growth was monitored with three weekly measurements until animals started to die.
- For the naked DNA variant in pAEC series in the case of mice immunized with 100 μg/animal, a decrease in tumor growth with respect to the negative control was observed (Table 4). In the variants included in the vector of the pMAE5Δ5 series with 5 CpG sites, independently of the VEGF isoform, tumor size was significantly reduced as compared to the negative control in the groups of mice immunized with doses of 10, 50, or 100 μg of DNA. In the case where the mutated variant pMAE5Δ5 VEGFKDR(−) was used, a significant reduction of tumor size was obtained at similar doses as those employed with the pMAE5Δ5-VEGF121.
- A survival analysis on day 43 evidenced a significant increase (with respect to the negative control) of the animals immunized with the variants pMAE5Δ5-VEGF121, pMAE5Δ5-VEGF165, pMAE5Δ5-VEGF189, and pMAE5Δ5 VEGFKDR(−), at doses of 50 and 100 μg per animal (Table 4).
-
TABLE 4 Tumor volume and survival in mice immunized with different variants of naked DNA containing the different isoforms of the VEGF gene and a mutated variant. [DNA Tumor Vol. (mm3) Survival Group μg] (Day 24) (Day 43) PAECΔ2-VEGF121 100 991.5 ± 354 (*) (ns) 50 1429.2 ± 396 (ns) (ns) 10 1506.6 ± 442 (ns) (ns) 1 1660.5 ± 456 (ns) (ns) PMAE5Δ5-VEGF121 100 645.0 ± 215 (***) (***) 50 850.1 ± 463 (***) (***) 10 992.1 ± 410 (*) (ns) 1 1560.3 ± 598 (ns) (ns) PMAE5Δ5-VEGF165 100 799.2 ± 335 (***) (***) 50 916.6 ± 390 (**) (**) 10 1000.5 ± 662 (*) (ns) 1 1845.3 ± 450 (ns) (ns) PMAE5Δ5-VEGF189 100 790.1 ± 235 (***) (***) 50 996.5 ± 255 (*) (**) 10 1050.2 ± 362 (*) (ns) 1 1670.2 ± 408 (ns) (ns) pMAE5Δ5 VEGFKDR(−) 100 550.1 ± 335 (***) (***) 50 894.7 ± 408 (**) (***) 10 991.8 ± 362 (*) (ns) 1 1489.3 ± 510 (ns) (ns) PBS pH 7.2 0 1673.9 ± 712 Note: Tumor volume is reported as mean ± standard deviation (SD) of the measures performed on the animals of each group, statistical comparisons were performed using one-way ANOVA and a Bonferroni post-test. In the case of survival, the reported statistical significance was obtained using the log-rank test to compare each group with respect to the control group, in the indicated day. Statistical signification is indicated as (ns) p ≦ 0.05 non-significant; (*) p ≦ 0.05; (**) p ≦ 0.01; and (***) p ≦ 0.001. - Groups of 20 mice were vaccinated or not with the following variants:
- 1. pMAE5Δ5-VEGF121 (50 μg of DNA/mouse) in PBS pH 7.2
2. pMAE5Δ5-KDR 1-3 (50 μg of DNA/mouse) in PBS pH 7.2
3. PBS pH 7.2 (Negative control) - In all cases immunization (day 0) was by im. route in the rear left foot with a total volume of 50 μl. All the animals were re-immunized 15 days later using the original immunization regime.
- On day 5 the induction of autoimmune arthritis began by immunization with chicken collagen type II (Sigma), a model previously described by Campbell et al. (Campbell I K et al Eur. J. Immunol. 30: 1568, 2000). This immunization was repeated on day 26. The four extremities of each mouse were evaluated on a daily basis according to the arthritis index that establishes punctuation from 0 to 3 for each limb due to the presence in the examination of signs of erythema (1), inflammation (2), or articular rigidity (3), with a maximal value of 12. Mice started to show clinical symptoms of arthritis development 23 days after the induction, with the higher incidences at 50 days. Table 5 shows the analysis of arthritis incidence in the animals of the different experimental groups. In days 40 and 55 a significant reduction on arthritis incidence was observed in vaccinated groups (1 and 2) as compared to control group.
-
TABLE 5 Incidence of arthritis on selected days (40 and 55). Group Incidence day 40 Incidence day 55 1 20/8 (40%) 20/9 (45%) 2 20/6 (30%) 20/12 (60%) 3 20/10 (50%) 20/14 (70%) - Groups of 15 mice were vaccinated or not with the following variants:
- 1. pMAE5Δ5-VEGF121 (50 μg of DNA/mouse) in PBS pH 7.2
2. pMAE5Δ5-KDR 1-3 (50 μg of DNA/mouse) in PBS pH 7.2
3. pMAE5 KDR C (50 μg/mouse) in PBS pH 7.2
4. PBS pH 7.2 (Negative control) - In every case, C57BI/6 mice were immunized by im. injection in the rear left foot with a total volume of 50 μl. All the animals were re-immunized 15 days later using the original immunization regime. Thirty days after the last immunization, the in vivo angiogenesis was evaluated in the animals using matrigel as described by Coughlin M C et al. (Coughlin M C et al. J. Clin. Invest. 101:1441, 1998). The animals previously vaccinated were divided in groups of 5 and injected subcutaneously in the abdominal middle line with 500 μl of matrigel (Becton Dickinson and Co., Franklin Lakes, N.J., USA) containing:
- 1. VEGF 50 ng/mL, Heparin 50 U/mL
2. 105 cells of B16-F10 melanoma - Six days later the animals were sacrificed and the matrigel plug was extracted. Hemoglobin contents in the plugs were analyzed according to the manufacturer instructions (Drabkin's reagent kit; Sigma Diagnostics Co., St. Louis, Mo., USA). Vaccination with the plasmids coding for VEGF or its receptor KDR inhibit significantly (p<0.001) the VEGF induced vascularization, as well as that induced by systems that are more complex: tumor cells.
- Different immunostimulatory agents, previously reported, were used, mixed with the pMAE5Δ5-VEGF121 construction following with the methodology described below. The Opc protein from the outer membrane of Neisseria meningitidis was purified according to the report of Musacchio et al. (Musacchio A et al. Vaccine, 67:751, 1997). 50 μg/mL of pMAE5Δ5-VEGF121 were added to 10 μg/mL of Opc with gentle shaking at acid pH. The resulting complex was extensively dialyzed overnight in endo-free PBS pH 7.2 (Sigma). The level of Opc protein-plasmid DNA association (Opc-pMAE5Δ5-VEGF121) was checked by DNA visualization using 1% agarose gel. More than 50% of the plasmid DNA was associated to the Opc protein.
- Very small particles (VSSP) coming from complex of outer membrane proteins (OMPC) of Neisseria meningitides, supplied by the Center of Molecular Immunology (R. Perez et al. U.S. Pat. Nos. 5,788,985, and 6,149,921), were used for combination with the plasmid DNA of interest. VSSP (1 mg) were incubated with 5 mg of pMAE5Δ5-VEGF121 overnight with gentle agitation. The resulting material was extensively dialyzed in endo-free PBS pH 7.2 (Sigma). The level of VSSP-plasmid DNA association (VSSP-pMAE5Δ5-VEGF121) was checked by DNA visualization using 1% agarose gel. More than 50% of the plasmid DNA was associated to the VSSP particles.
- The Hepatitis C and Hepatitis B core particulated antigens (HCcAg and HBcAg) were produced according to a previous report (Lorenzo L J et al., Biochem Biophys Res Commun 281:962, 2001). One mg of the antigens were mixed with 5 mg of the plasmid in an overnight incubation. The levels of HCcAg or HBcAg-plasmid DNA association (HCcAg-pMAE5Δ5-VEGF121, and HBcAg-pMAE5Δ5-VEGF121, respectively) were checked by DNA visualization using 1% agarose gel. More than 50% of the DNA was associated to the antigenic particle, in each case.
- Groups of 10 C57BL6 mice were vaccinated or not with the following variants:
- 1. pMAE5Δ5-VEGF121 (1, 10 and 50 μg of DNA/mouse) in PBS pH 7.2
- 6. PBS pH 7.2 (Negative control for group 1)
7. Opc (Negative control for group 2)
8. VSSP (Negative control for group 3)
9. HBcAg (Negative control for group 4)
10. HCcAg (Negative control for group 5) - Immunization procedures, as well as tumor challenge and tumor volume measurements were similar to those described in the previous example. The vaccine variants with doses similar or higher to 10 μg of DNA/mouse decreased tumor growth in comparison to the respective negative controls (Table 6). A significant superior survival as compared to that of the respective control, was observed for the animals immunized with the VEGF gene, associated or not with Opc, VSSP, HCcAg and HBcAg, as immunopotentiator vehicles. All the variants with vehicle showed a significant superior survival versus the respective control, for doses starting with 10 μg/mouse, while the naked DNA variant with the vector pMAE5Δ5-VEGF121, resulted significantly different from the negative control at the dose of 50 μg/mouse (Table 6).
-
TABLE 6 Tumor volume and survival of mice immunized using different immunostimulatory agents. [DNA Tumor Vol. (mm3). Survival Group μg] (Day 24) (Day 43) pMAE5Δ5-VEGF 50 1050.9 ± 689 (**) (ns) 10 1229.0 ± 596 (*) (ns) 1 1895.3 ± 596 (ns) (ns) OpC-pMAE5Δ5-VEGF 50 960.6 ± 456 (**) (**) 10 1100.5 ± 615 (**) (*) 1 1654.8 ± 663 (ns) (ns) VSSP-pMAE5Δ5-VEGF 50 884.6 ± 410 (***) (**) 10 1002.3 ± 598 (**) (*) 1 1532.7 ± 745 (ns) (ns) HBcAg-pMAE5Δ5-VEGF 50 950.1 ± 570 (**) (**) 10 1230.5 ± 662 (*) (*) 1 1867.2 ± 652 (ns) (ns) HCcAg-pMAE5Δ5-VEGF 50 950.1 ± 570 (**) (**) 10 1230.5 ± 662 (*) (*) 1 1867.2 ± 652 (ns) (ns) OpC (5 μg/mouse/dose) 5 μg 2059.0 ± 687 (ns) (ns) VSSP 2156.0 ± 759 (ns) (ns) HBcAg (5 μg/mouse/dose) 1998.2 ± 798 (ns) (ns) HCcAg (5 μg/mouse/dose) 1897.0 ± 812 (ns) (ns) PBS pH 7.2 2073.0 ± 816 (ns) (ns) Note: Tumor volume is reported as mean ± standard deviation (SD) of the measures performed on the animals of each group, statistical comparisons were performed using one-way ANOVA and a Bonferroni post-test. In the case of survival, the reported statistical significance was obtained using the log-rank test to compare each group with respect to the control group, in the indicated day. Statistical signification is indicated as (ns) p ≦ 0.05 non-significant; (*) p ≦ 0.05; (**) p ≦ 0.01; and (***) p ≦ 0.001. - Groups 10 C57BL6 mice were vaccinated or not with the following variants:
- VEGF165 (20 μg/mouse) with Complete and Incomplete Freund adjuvant Complete and Incomplete Freund adjuvant (negative control)
- VEGF165 antigen was obtained from a commercial source (SIGMA) with more than 97% purity. Mice were immunized by the intraperitoneal route using Complete Freund's adjuvant (Sigma) with re-immunizations in days 15 and 30 by the same route but using Incomplete Freund's adjuvant. Tumor challenge, and measurements of tumor volume were similar to those described in the previous example.
- A significant reduction in tumor volume and increase survival were observed in the VEGF immunized group as compared to the control non-immunized group. The effect was similar to those found in previous experiments using VEGF DNA.
- C57BL6 mice were immunized or not with doses of 50 μg of pMAE5Δ5-VEGF121 DNA/mouse using the procedures described in the example 5. Mice were sacrificed at 45 days after first immunization. CD8+, CD4+ and B-lymphocytes of these mice were separated using magnetic beads (Dynabeads, USA), according to the manufacturer instructions.
- Groups of 10 six-week old C57BL6 SCID mice were reconstituted with the following combinations of the previously extracted lymphocytes.
- Group 1: CD8+ T-lymphocytes and CD4+ T-lymphocytes from mice immunized with pMAE5Δ5-VEGF121 DNA. B-lymphocytes were not reconstituted.
- Group 2: B-lymphocytes and CD4+ T-lymphocytes from immunized mice, and CD8+ T-lymphocytes from non-immunized mice.
- Group 3: B-lymphocytes, CD8+ T-lymphocytes and CD4+ T-lymphocytes from immunized mice, as a positive control of the experiment.
- Group 4: B-lymphocytes, CD8+ T-lymphocytes, and CD4+ T-lymphocytes from non-immunized mice, as a negative control of the experiment.
- Reconstituted SCID mice were challenged sc. with 104 B16-F10 melanoma cells. Tumor growth was monitored by three weekly measurements until mice start to die. Anti-VEGF antibody levels were analyzed through a laboratory ELISA. 96-well plates were incubated overnight with a 0.5 μg/ml solution of VEGF165 (Sigma). The wells were blocked with PBS-BSA 1% (BDH, UK) solution, and later incubated with serial dilutions of the animal sera. After washing with PBS-Tween 0.05%, a commercially available polyclonal anti mouse IgG (Sigma, A0168) was added. The signal was amplified in the presence of the commercial substrate ortho-phenilene-diamine (OPD, Sigma).
- Table 7 reflects the results of tumor volume (Day 24) and survival (Day 40) of the groups of mice submitted to tumor challenge. Beginning on the day 15 after reconstitution, the animals of the groups 1 to 3 experienced a reduction in tumor size as compared to group 4, reconstituted with lymphocytes from non-immunized mice. Thus, the effect that provokes the immune system in the immunized mice, that allows the reduction in tumor size, is related to humoral and cellular responses, being the last one of the cytotoxic type (CTL), due to the absence of anti-VEGF antibodies in group 1. Nevertheless, in the experimental conditions used survival only increased in group 3 (B and T lymphocytes of immunized mice), as compared to the rest of the groups (Table 7). In the partially reconstituted animals where B or T of the CTL type responses were absent (groups 1 and 2, respectively) the survival was not different from the negative control. These results demonstrate that the combination of humoral and cellular responses (group 4), have a synergic effect that enables an effective response able to prolong the survival of mice submitted to the tumor challenge.
-
TABLE 7 Tumor volume and survival in SCID mice reconstituted with lymphocytes from pMAE5Δ5-VEGF121 immunized mice. Mice donating lymphocytes to the C57BL/6 SCID Tumor Vol. Survival Group B Lymph. CD4+ Lymph. CD8+ Lymph. (Day 24) (Day 40) 1 — immunized immunized 1067.8 ± 689 (ns) (ns) 2 immunized immunized non immunized 1129.0 ± 596 (ns) (ns) 3 immunized immunized immunized 652.3 ± 396 (***) (***) 4 Non immunized Non immunized Non immunized 1856.0 ± 756 — Note: Donor mice were immunized or not with doses of 50 μg of pMAE5Δ5-VEGF DNA per mouse. Tumor volume is reported as mean ± standard deviation (SD) of the measures performed on the animals of each group, statistical comparisons were performed using one-way ANOVA and a Bonferroni post-test. In the case of survival, the reported statistical significance was obtained using the log-rank test to compare each group with respect to the control group, in the indicated day. Statistical signification is indicated as (ns) p ≦ 0.05 non-significant; (*) p ≦ 0.05; (**) p ≦ 0.01; and (***) p ≦ 0.001. - Groups of 15 C57BL6 female mice were injected by im. route with the following variants:
- 1. pMAE5Δ5-VEGF121 (50 μg/mouse) in PBS pH 7.2
- In every case, mice were immunized by im. injection in the rear left foot with a total volume of 50 μl. All the animals were re-immunized 15 days later using the original immunization regime. Thirty days after the last immunization 5 randomly selected animals from each group were sacrificed to analyze the immunological state of the immunized and control animals, as well as the toxicity of vaccination on organs and tissues, through macroscopic and histological evaluations.
- Remaining animals of each group received a sc injection of 104 melanoma B16-F10 cells in the right ventral zone. At 15 and 30 days after tumor cells injection, 5 mice per group were sacrificed and evaluated as previously described.
- Toxic events were not evidenced at macroscopic level in any of the evaluated animals, and histopathological analysis reveal no damage in any of the organs examined 30 days after the last immunization. Immunological evaluation consisted of: (1) evaluation of murine VEGF levels in serum; (2) cellular content of T and B lymphocytes, as well as the degree of maturity of dendritic cells in spleen, and in brachial axillary and inguinal lymph nodes.
- The analysis of the levels of murine VEGF (R&D kit for murine VEGF) in the sera of un-treated animals showed that with the increase of time of exposal to tumor, the VEGF levels increased in serum, in concordance with the increase of tumor size with time. In the group immunized against human VEGF a significant reduction (p<0.001 ANOVA, post-test Bonferroni) of murine VEGF levels was detected, that lasted past 30 days after the tumor challenge.
- The status of the immune system of the animals sacrificed on each moment was analyzed through the study of the proportions of the cellular populations present on lymph nodes and spleen, according to the reports of Gabrilovich et al. (Gabrilovich D et al. Blood 92:4150, 1998). For theses studies, commercial monoclonal antibodies that recognize CD3, CD19, CD11c and CD86 (B7-2) molecules (Pharmingen) labeled with fluorescein isothiocyanate (FITC) and phycoerythrine (PE), were used, that allowed the visualization of the cellular populations using a flow cytometer (FACS). Results obtained are shown in table 8.
-
TABLE 8 Summary of the results of FACS analysis of cell populations according to surface markers. Fraction enriched with dendritic Total of Cells cells Lymph Nodes Spleen Lymph Nodes Spleen Group (day) CD-19 CD-3 CD-19 CD-3 CD-11C/B7-2 CD-11C/B7-2 A. Non immunized Non immunized 8% 86% 38.1% 40.8% 60% 62.4% (30 Days) After tumor 20.1% 60.5% 3.8 11.4% 32.8% 10.2% challenge (60 Days) B. Immunized Immunized 7.2% 87.3% 40% 39% 58.6% 60.3% (30 Days) After tumor 10.9% 80.1% 25.4 34% 53.5% 52.9% challenge (60 Days) Note: In every case, values indicate the percent of positive cells from the total of quantified cells. - The analyses of lymphoid cell populations and of the maturation of dendritic cells in the animals, 30 days after the immunization, indicate that the vaccination with the VEGF DNA does not induce any change in the immune status of the animal. Nevertheless, 30 days after the tumor implantation, the non-vaccinated animals show a decrease in the T-lymphocyte/B-lymphocyte ratio (CD3/CD19) both in lymph nodes and in spleen, with respect to the ratio before the tumor challenge. Furthermore, in particular in the spleen, there is a significant reduction in the number of lymphoid cells. A reduction in the number of mature dendritc cells both in lymph nodes and in spleen was also observed in these animals. In the group of mice vaccinated with the VEGF DNA a significant recovery in all parameters was evidenced, that could be correlated with the reduction of the VEGF levels in the sera observed in the animals of this group.
Claims (3)
1.-33. (canceled)
34. An immunogenic composition comprising a fusion protein containing a VEGFR2 or fragments thereof and a mutant of VEGF, administered in the presence of or incorporated into Neisseria meningitidis outer membrane derived VSSP.
35.-79. (canceled)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/787,522 US20080254050A1 (en) | 2002-04-15 | 2007-04-17 | Antiangiogenic active immunotherapy |
US12/612,455 US8883724B2 (en) | 2002-04-15 | 2009-11-04 | Active antiangiogenic therapy |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CUCU2002/0076 | 2002-04-15 | ||
CU20020076A CU23178A1 (en) | 2002-04-15 | 2002-04-15 | ANTIANGIOGEN ACTIVE ACTIVE IMMUNOTHERAPY |
US10/511,384 US7556809B2 (en) | 2002-04-15 | 2003-04-11 | Active antiangiogenic therapy |
PCT/CU2003/000004 WO2003086450A1 (en) | 2002-04-15 | 2003-04-11 | Active antiangiogenic therapy |
US11/787,522 US20080254050A1 (en) | 2002-04-15 | 2007-04-17 | Antiangiogenic active immunotherapy |
Related Parent Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10511384 Division | 2003-04-11 | ||
PCT/CU2003/000004 Division WO2003086450A1 (en) | 2002-04-15 | 2003-04-11 | Active antiangiogenic therapy |
US10/511,384 Division US7556809B2 (en) | 2002-04-15 | 2003-04-11 | Active antiangiogenic therapy |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/612,455 Division US8883724B2 (en) | 2002-04-15 | 2009-11-04 | Active antiangiogenic therapy |
Publications (1)
Publication Number | Publication Date |
---|---|
US20080254050A1 true US20080254050A1 (en) | 2008-10-16 |
Family
ID=40134850
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/511,384 Expired - Lifetime US7556809B2 (en) | 2002-04-15 | 2003-04-11 | Active antiangiogenic therapy |
US11/787,522 Abandoned US20080254050A1 (en) | 2002-04-15 | 2007-04-17 | Antiangiogenic active immunotherapy |
US12/612,455 Active 2025-07-07 US8883724B2 (en) | 2002-04-15 | 2009-11-04 | Active antiangiogenic therapy |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/511,384 Expired - Lifetime US7556809B2 (en) | 2002-04-15 | 2003-04-11 | Active antiangiogenic therapy |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/612,455 Active 2025-07-07 US8883724B2 (en) | 2002-04-15 | 2009-11-04 | Active antiangiogenic therapy |
Country Status (17)
Country | Link |
---|---|
US (3) | US7556809B2 (en) |
EP (1) | EP1502599B1 (en) |
JP (1) | JP4741800B2 (en) |
KR (1) | KR100704127B1 (en) |
CN (1) | CN1646153B (en) |
AR (1) | AR039285A1 (en) |
AT (1) | ATE511851T1 (en) |
AU (1) | AU2003218603B2 (en) |
CA (1) | CA2480079C (en) |
CU (1) | CU23178A1 (en) |
DK (1) | DK1502599T3 (en) |
ES (1) | ES2365329T3 (en) |
PT (1) | PT1502599E (en) |
RU (1) | RU2329824C2 (en) |
SI (1) | SI1502599T1 (en) |
WO (1) | WO2003086450A1 (en) |
ZA (1) | ZA200408460B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090252752A1 (en) * | 2002-09-12 | 2009-10-08 | Oncotherapy Science, Inc. | Kdr peptides and vaccines comprising the same |
US8975229B2 (en) | 2009-06-11 | 2015-03-10 | Oncotherapy Science, Inc. | Methods for treating a disease caused by choroidal neovascularization |
Families Citing this family (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CU23178A1 (en) * | 2002-04-15 | 2006-09-22 | Ct Ingenieria Genetica Biotech | ANTIANGIOGEN ACTIVE ACTIVE IMMUNOTHERAPY |
EP1804835B9 (en) | 2004-09-13 | 2012-05-02 | Genzyme Corporation | Multimeric constructs |
SI2289533T1 (en) | 2005-02-28 | 2013-11-29 | Oncotherapy Science, Inc. | Epitope peptides derived from vascular endothelial growth factor receptor 1 and vaccines containing these peptides |
US20060234941A1 (en) * | 2005-04-15 | 2006-10-19 | The Gov. Of The Usa As Represented By The Secretary Of The Dept. Of Health & Human Services | Peptide epitopes of VEGFR-2/KDR that inhibit angiogenesis |
TWI428143B (en) | 2006-01-18 | 2014-03-01 | Gen Hospital Corp | Methods of increasing lymphatic function |
CN101394857A (en) * | 2006-02-01 | 2009-03-25 | 爱吉恩公司 | Composite for treating cancer containing oligonucleotide and nontoxic LPS |
ES2405763T3 (en) * | 2007-02-16 | 2013-06-03 | Oncotherapy Science, Inc. | Vaccination for choroidal neovascularization |
US20120028880A1 (en) * | 2007-08-08 | 2012-02-02 | Renata Pasqualini | Vegfr-1/nrp-1 targeting peptides |
TWI436775B (en) * | 2007-08-24 | 2014-05-11 | Oncotherapy Science Inc | Combination therapy for pancreatic cancer using an antigenic peptide and chemotherapeutic agent |
CN102046807B (en) * | 2008-04-24 | 2014-11-26 | 3M创新有限公司 | Analysis of nucleic acid amplification curves using wavelet transformation |
US20110293625A1 (en) * | 2008-11-21 | 2011-12-01 | Krishna Addepalli Murali | Inhibition of vegf-a secretion, angiogenesis and/or neoangiogenesis by sina mediated knockdown of vegf-c and rhoa |
DK2403935T3 (en) | 2009-03-04 | 2017-09-11 | Univ Pennsylvania | COMPOSITIONS CONTAINING ANGIOGENIC FACTORS AND METHODS OF USE THEREOF |
ES2652508T3 (en) | 2010-08-06 | 2018-02-02 | Genzyme Corporation | VEGF antagonist compositions and their uses |
RU2444378C1 (en) * | 2010-09-06 | 2012-03-10 | Олег Германович Макеев | Method of treating coronary insufficiency in simulated myocardial ischemia |
WO2012088563A1 (en) * | 2010-11-24 | 2012-07-05 | Vegenics Pty Limited | Vegfr-2-specific forms of vegf-d and vegf-c and uses thereof |
WO2013074820A1 (en) * | 2011-11-16 | 2013-05-23 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Immunogenic tumor associated stromal cell antigen peptides and methods of their use |
CN104768575B (en) * | 2012-08-31 | 2017-09-08 | 国立大学法人大阪大学 | DNA vaccination containing VEGF specificity epitopes and/or ANG2 specificity epitope |
US20160331807A1 (en) * | 2014-01-21 | 2016-11-17 | University Of Helsinki | Therapeutic use of vegfr-3 ligands |
KR101509047B1 (en) * | 2014-07-28 | 2015-04-08 | 한국생명공학연구원 | Method for diagnosing and screening asthma using Neuropilin 1 |
EP3402815B1 (en) | 2016-01-15 | 2023-12-06 | University of Maryland, College Park | Endo-s2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins |
EP3486254A4 (en) * | 2016-07-05 | 2019-12-18 | Ibentrus, Inc. | Cancer treatment composition for inhibiting tumor angiogenesis, containing vegf deep blocker, and preparation method therefor |
EP3497229A1 (en) | 2016-08-10 | 2019-06-19 | University of Maryland, College Park | Designer alpha 1,6-fucosidase mutants enable direct core fucosylation of intact n-glycopeptides and n-glycoproteins |
CN110612115A (en) * | 2017-05-19 | 2019-12-24 | 学校法人庆应义塾 | Peptide vaccine and peptide vaccine composition against cranial nerve disease |
US11459380B2 (en) | 2017-06-29 | 2022-10-04 | University Of Maryland, College Park | Transglycosylation of endo-S and endo-S mutants for antibody glycosylation remodeling |
WO2020151666A1 (en) * | 2019-01-25 | 2020-07-30 | 四川大学华西医院 | Biomarker for hemangioma treament |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6149921A (en) * | 1993-12-29 | 2000-11-21 | Centro De Inmunologia Molecular | Vaccine compositions for eliciting an immune response against N-acetylated gangliosides and their use for cancer treatment |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5219739A (en) * | 1989-07-27 | 1993-06-15 | Scios Nova Inc. | DNA sequences encoding bVEGF120 and hVEGF121 and methods for the production of bovine and human vascular endothelial cell growth factors, bVEGF120 and hVEGF121 |
CA2158745C (en) * | 1993-03-25 | 2007-06-19 | Richard L. Kendall | Inhibitor of vascular endothelial cell growth factor |
AU3374795A (en) * | 1994-08-29 | 1996-03-22 | Prizm Pharmaceuticals, Inc. | Conjugates of vascular endothelial growth factor with targeted agents |
EP1032583A4 (en) * | 1998-03-06 | 2005-02-02 | Imclone Systems Inc | Active immunization against angiogenesis-associated antigens |
EP1075184A4 (en) * | 1998-05-08 | 2002-01-30 | Sloan Kettering Inst Cancer | Compositions and methods for active vaccination |
EP1156824A2 (en) * | 1999-03-11 | 2001-11-28 | EntreMed, Inc. | Compositions and methods for treating cancer and hyperproliferative disorders |
AU2001228742A1 (en) * | 2000-02-04 | 2001-08-14 | Supratek Pharma, Inc. | Ligand for vascular endothelial growth factor receptor |
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 |
-
2002
- 2002-04-15 CU CU20020076A patent/CU23178A1/en unknown
-
2003
- 2003-04-10 AR ARP030101264A patent/AR039285A1/en unknown
- 2003-04-11 CA CA2480079A patent/CA2480079C/en not_active Expired - Lifetime
- 2003-04-11 PT PT03711811T patent/PT1502599E/en unknown
- 2003-04-11 EP EP03711811A patent/EP1502599B1/en not_active Expired - Lifetime
- 2003-04-11 WO PCT/CU2003/000004 patent/WO2003086450A1/en active Application Filing
- 2003-04-11 AT AT03711811T patent/ATE511851T1/en active
- 2003-04-11 KR KR1020047016584A patent/KR100704127B1/en active IP Right Grant
- 2003-04-11 US US10/511,384 patent/US7556809B2/en not_active Expired - Lifetime
- 2003-04-11 SI SI200332037T patent/SI1502599T1/en unknown
- 2003-04-11 JP JP2003583468A patent/JP4741800B2/en not_active Expired - Fee Related
- 2003-04-11 RU RU2004133338/15A patent/RU2329824C2/en active IP Right Revival
- 2003-04-11 DK DK03711811.4T patent/DK1502599T3/en active
- 2003-04-11 CN CN03808567.4A patent/CN1646153B/en not_active Expired - Lifetime
- 2003-04-11 AU AU2003218603A patent/AU2003218603B2/en not_active Ceased
- 2003-04-11 ES ES03711811T patent/ES2365329T3/en not_active Expired - Lifetime
-
2004
- 2004-10-19 ZA ZA200408460A patent/ZA200408460B/en unknown
-
2007
- 2007-04-17 US US11/787,522 patent/US20080254050A1/en not_active Abandoned
-
2009
- 2009-11-04 US US12/612,455 patent/US8883724B2/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6149921A (en) * | 1993-12-29 | 2000-11-21 | Centro De Inmunologia Molecular | Vaccine compositions for eliciting an immune response against N-acetylated gangliosides and their use for cancer treatment |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090252752A1 (en) * | 2002-09-12 | 2009-10-08 | Oncotherapy Science, Inc. | Kdr peptides and vaccines comprising the same |
US7695720B2 (en) | 2002-09-12 | 2010-04-13 | Oncotherapy Science, Inc. | KDR peptides and vaccines comprising the same |
US20100215676A1 (en) * | 2002-09-12 | 2010-08-26 | Oncotherapy Science, Inc. | Kdr peptides and vaccines comprising the same |
US8206719B2 (en) | 2002-09-12 | 2012-06-26 | Oncotherapy Science, Inc. | KDR peptides and vaccines comprising the same |
US8574585B2 (en) | 2002-09-12 | 2013-11-05 | Oncotherapy Science, Inc. | KDR peptides and vaccines comprising the same |
US8574586B2 (en) | 2002-09-12 | 2013-11-05 | Oncotherapy Science, Inc. | KDR peptides and vaccines comprising the same |
US8975229B2 (en) | 2009-06-11 | 2015-03-10 | Oncotherapy Science, Inc. | Methods for treating a disease caused by choroidal neovascularization |
Also Published As
Publication number | Publication date |
---|---|
US8883724B2 (en) | 2014-11-11 |
EP1502599B1 (en) | 2011-06-08 |
KR20040111526A (en) | 2004-12-31 |
AR039285A1 (en) | 2005-02-16 |
KR100704127B1 (en) | 2007-04-05 |
RU2329824C2 (en) | 2008-07-27 |
ZA200408460B (en) | 2005-08-30 |
CN1646153B (en) | 2017-05-24 |
US7556809B2 (en) | 2009-07-07 |
SI1502599T1 (en) | 2011-10-28 |
CA2480079A1 (en) | 2003-10-23 |
CA2480079C (en) | 2013-07-30 |
JP2006501145A (en) | 2006-01-12 |
US20100047265A1 (en) | 2010-02-25 |
CU23178A1 (en) | 2006-09-22 |
WO2003086450A1 (en) | 2003-10-23 |
JP4741800B2 (en) | 2011-08-10 |
DK1502599T3 (en) | 2011-08-01 |
PT1502599E (en) | 2011-08-03 |
AU2003218603A1 (en) | 2003-10-27 |
EP1502599A1 (en) | 2005-02-02 |
ATE511851T1 (en) | 2011-06-15 |
CN1646153A (en) | 2005-07-27 |
ES2365329T3 (en) | 2011-09-29 |
RU2004133338A (en) | 2005-06-10 |
US20050175624A1 (en) | 2005-08-11 |
AU2003218603B2 (en) | 2009-05-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8883724B2 (en) | Active antiangiogenic therapy | |
US10682402B2 (en) | MSI-specific frameshift peptides (FSP) for prevention and treatment of cancer | |
US8425897B2 (en) | Compositions containing LAG-3 and cells that secrete GM-CSF and methods of use | |
EP1238086B1 (en) | Vaccine for enhancing immune responses to herpes simplex virus | |
KR101810840B1 (en) | Msi-specific framshift peptides (fsp) for prevention and treatment of cancer | |
EP2572725A1 (en) | MSI-specific frameshift peptides (FSP) for prevention and treatment of cancer | |
US6867000B2 (en) | Method of enhancing immune responses to herpes | |
JP5890769B2 (en) | MSI-specific frameshift peptides (FSP) for cancer prevention and treatment | |
JP2016028025A (en) | Msi-specific frameshift peptides (fsp) for prevention and treatment of cancer | |
BRPI0400755A2 (en) | use of polypeptides and / or oligonucleotides, and vaccine composition | |
CA2799803C (en) | Msi-specific frameshift peptides (fsp) for prevention and treatment of cancer | |
AU2016201986A1 (en) | MSI-specific Frameshift Peptides (FSP) for Prevention and Treatment of Cancer | |
JP2009541235A (en) | Use of PPD for the adjuvant action of nucleic acid vaccines |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |