IE893548L - Neoglycoproteins, the preparation and use thereof - Google Patents
Neoglycoproteins, the preparation and use thereofInfo
- Publication number
- IE893548L IE893548L IE893548A IE354889A IE893548L IE 893548 L IE893548 L IE 893548L IE 893548 A IE893548 A IE 893548A IE 354889 A IE354889 A IE 354889A IE 893548 L IE893548 L IE 893548L
- Authority
- IE
- Ireland
- Prior art keywords
- neoglycoprotein
- spdp
- sugar
- hsa
- disialolactose
- Prior art date
Links
- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 24
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims description 7
- 238000005859 coupling reaction Methods 0.000 claims abstract description 21
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 18
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 18
- 230000008878 coupling Effects 0.000 claims abstract description 18
- 238000010168 coupling process Methods 0.000 claims abstract description 18
- 229960005486 vaccine Drugs 0.000 claims abstract description 9
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 8
- 210000003022 colostrum Anatomy 0.000 claims abstract description 8
- 235000021277 colostrum Nutrition 0.000 claims abstract description 8
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 235000000346 sugar Nutrition 0.000 claims description 23
- 150000002270 gangliosides Chemical class 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- 241000283690 Bos taurus Species 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 229940039227 diagnostic agent Drugs 0.000 claims description 3
- 239000000032 diagnostic agent Substances 0.000 claims description 3
- 230000002860 competitive effect Effects 0.000 claims description 2
- 238000003127 radioimmunoassay Methods 0.000 claims description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 10
- 102000004856 Lectins Human genes 0.000 abstract description 5
- 108090001090 Lectins Proteins 0.000 abstract description 5
- 239000002523 lectin Substances 0.000 abstract description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 239000003446 ligand Substances 0.000 abstract description 3
- 230000004807 localization Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 238000002560 therapeutic procedure Methods 0.000 abstract description 3
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 238000001212 derivatisation Methods 0.000 description 11
- WHMDPDGBKYUEMW-UHFFFAOYSA-N pyridine-2-thiol Chemical compound SC1=CC=CC=N1 WHMDPDGBKYUEMW-UHFFFAOYSA-N 0.000 description 10
- 229920005654 Sephadex Polymers 0.000 description 9
- 239000012507 Sephadex™ Substances 0.000 description 9
- 230000008033 biological extinction Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000012064 sodium phosphate buffer Substances 0.000 description 6
- 150000008163 sugars Chemical class 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 5
- 230000008030 elimination Effects 0.000 description 5
- 238000003379 elimination reaction Methods 0.000 description 5
- GIVLTTJNORAZON-HDBOBKCLSA-N ganglioside GM2 (18:0) Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 GIVLTTJNORAZON-HDBOBKCLSA-N 0.000 description 5
- 201000001441 melanoma Diseases 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 4
- 150000002339 glycosphingolipids Chemical class 0.000 description 4
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- SKIUVOVOIJBJPN-UHFFFAOYSA-N 4,5-dimethoxybenzene-1,2-diamine Chemical compound COC1=CC(N)=C(N)C=C1OC SKIUVOVOIJBJPN-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- -1 for example Proteins 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000002731 protein assay Methods 0.000 description 3
- 238000006268 reductive amination reaction Methods 0.000 description 3
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 3
- CERZMXAJYMMUDR-QBTAGHCHSA-N 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid Chemical compound N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO CERZMXAJYMMUDR-QBTAGHCHSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 206010003571 Astrocytoma Diseases 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241001467552 Mycobacterium bovis BCG Species 0.000 description 2
- SQVRNKJHWKZAKO-LUWBGTNYSA-N N-acetylneuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-LUWBGTNYSA-N 0.000 description 2
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229940106189 ceramide Drugs 0.000 description 2
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940060155 neuac Drugs 0.000 description 2
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000011877 solvent mixture Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 1
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 241001222774 Salmonella enterica subsp. enterica serovar Minnesota Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 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
-
- 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
- A61K39/385—Haptens or antigens, bound to carriers
-
- 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
- A61K39/001169—Tumor associated carbohydrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Hematology (AREA)
- Mycology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The synthesis and use of neoglycoproteins is described. Monosialolactose and disialolactose are isolated from cow colostrum and coupled using a coupling reagent to human serum albumin (HSA) as carrier protein. These neoglycoproteins are suitable as vaccines and as ligands for the localisation and therapy of lectin-expressing tumour tissues.
Description
- 1 - "7 > ~r s / o 0 I Z The invention relates to the synthesis and use of neo-glycoproteins. Monosialolactose and disialolactose are isolated from bovine colostrum and coupled by means of a coupling reagent to human serum albumin (HSA) as carrier 5 protein. The neoglycoproteins are suitable as vaccines and, moreover, as ligands for the localization and therapy of lectin-expressing tumor tissues.
Gangliosides, i.e. glycosphingolipids which contain sialic acid, are plasma membrane lipids composed of a 10 hydrophobic ceramide portion and a hydrophilic carbohydrate portion. Glycosphingolipids are anchored with the ceramide portion, which is composed of sphingosine and a long-chain fatty acid, in the outer plasma membrane in such a way that the oligosaccharide chains project into 15 the extracellular space. Because of the fact that they form on the cell .surface cell-differentiating and species-specific patterns, it is thought that they have a biologically important function in cellular events.
Glycosphingolipids are typical of all mammalian cells but 2 0 usually occur in only small amounts. Glycosphingolipids which contain sialic atid, i.e. gangliosides, are particularly concentrated on neuronal plasma membranes. Gangliosides are also strongly expressed on the cell surfaces of tumors of neuroectodermal origin (such as 25 melanoma, astrocytoma, neuroblastoma etc.). Some of these gangliosides are ones which occur on normal mammalian cells, but also in the brain and on nerve fibers, only in relatively small amounts. This is why the relevant gangliosides GM2, GM3, GD2 and GD3 have become interest- 3 0 ing as tumor-associated antigens. The possibilities of a tumor immunotherapy of tumors of neuroectodermal origin with these gangliosides as target antigen are also being investigated by various research groups. Two different approaches have been followed to date with some success: 35 1. The use of of monoclonal antibodies directed against the appropriate gangliosides and - 2 - 2. active immunization with the appropriate gangliosides.
Investigations have shown that antibodies against purified GM2 ganglioside cannot be raised in melanoma pa-5 tients. Not until adjuvants such as Bacillus Calmette-Guerin (BCG) or Salmonella minnesota were used, and the . patients were pretreated with cyclophosphamide, were IgM antibodies induced against ganglioside GM2 (Livingston et al. (1987), Proc. Natl. Acad. Sci. 84, 2911-2915). 10 Moreover, a whole-cell vaccine composed of human or murine melanoma or astrocytoma cell lines having a high GM2 ganglioside content have been injected into melanoma patients and resulted in induction of anti-GM2 antibodies of the IgM isotope (Livingston et al. loc. cit.). 15 Studies to date on melanoma patients have shown that an elevated titer of antibodies against ganglioside GM2 correlates with an increased survival time.
The central problems in the design of a ganglioside vaccine are the poor immunogenicity of the purified 20 gangliosides and the difficulty of isolating them.
Such difficulties also exist in general in the investigation of sugar-binding proteins. This is the reason, for example, for the development of neoglycopro-teins containing a simple sugar such as, for example, 25 galactose and a carrier protein such as, for example, bovine serum albumin (BSA) in order to analyze the binding of lectins better [J. Histochem. and Cytochem. (1984), 32 (10), pp. 1091-1098].
One approach to increasing the immunogenic ity of the 3 0 ganglioside GM2 is to be found in U.S. Patent No. 4,557,931. This discloses a neoglycoprotein whose sugar portion derives from GM2 and whose protein portion is HSA. This oligosaccharide-HSA conjugate is used as vaccine for inducing antibodies which react with GM2, and - 3 as diagnostic agent.
It was also known that the sugar portions of gangliosides GM3 and GD3, monosialolactose and disialolactose respectively, can easily be obtained from bovine colostrum. We 5 have found, surprisingly, that monosialolactose and disialolactose, coupled to HSA by means of a coupling reagent, react with monoclonal antibodies which have been raised against gangliosides GM3 and GD3 respectively. Conversely, the abovementioned neoglycoproteins are able 10 to raise antibodies which react with gangliosides GM3 and GD3 and are therefore suitable as vaccines for specific tumor immunotherapy. Furthermore, they are also suitable, for example, as ligands for the localization and therapy of lectin-expressing tumor tissues. 15 Accordingly, the invention relates to a) neoglycoproteins composed of monosialo- or disialolactose, with carrier proteins, including enzymes, preferably human serum albumin, b) a process for the preparation thereof, the preferred 2 0 starting material being bovine colostrum as source of sugar, c) and the use thereof as vaccines or as diagnostic aids, for example for detecting gangliosides in competitive radioimmunoassays as part of tumor 25 diagnosis as well as therapeutic or diagnostic agents for lectin-expressing tumors.
The invention is further contained in the examples and the patent claims. - 4 - Examples 1. Isolation of monosialolactose and disialolactose from bovine colostrum Bovine colostrum was worked up by the method of v. 5 Nicolai, with minor modifications (v. Nicolai, Thesis, Bonn University, 1971).
Bovine colostrum (10 liters) was centrifuged to remove fats, and proteins were precipitated with 50 % acetone in the cold. After renewed centrifugation the supernatant 10 was concentrated in a rotary evaporator to 1/10 of the original volume.
The crude fraction was passed through a Sephadex G-15 column to remove salts, and the sugar-positive and chloride-negative fractions were combined and concentrat-15 ed. The individual sugars were separated by ion exchange on a DEAE-Sephadex A-25 column in 0.1 M Tris/HCl buffer, pH 7.5, using a sodium chloride gradient. The appropriate sugar fractions were combined, concentrated and passed through a Sephadex G-15 column to remove salts. 20 Sugars were detected in the individual fractions from the column working-up steps by the Ehrlich and anthrone tests or by fractionation by thin-layer chromatography on silica gel plates using the solvent mixture pyridine/ ethyl acetate/glacial acetic acid/water (6:3:1:3) and 25 spraying the plate with resorcinol. 2. Coupling of monosialolactose and disialolactose to human serum albumin (HSA) by means of the coupling reagent SPDP There are several possibilities in principle for the 30 coupling of the sugars to protein. The sugars can, for example, be coupled directly to the free epsiIon-amino-lysyl groups of the protein, or else via homo- or hetero- - 5 - bifunctional coupling reagents.
The coupling reactions using N-succinimidyl 3-(2-pyridyl-dithio)propionate (SPDP) as coupling reagent are described hereinafter. The reaction steps substantially 5 correspond to the published method (J. Carlsson et al. (1978), Biochem. J. 173, 723-737). a) Reductive amination of monosialolactose and disialolactose The method of Wiegandt was used for the redutive amina-10 tion at the Cx atom of the glucose in monosialolactose and disialolactose in methanolic ammonium acetate solution with the addition of sodium cyanoborohydride (H. Wiegandt, W. Ziegler, (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 11-18). 15 The reductively aminated sugar derivative was purified on a Biogel P2 column.
Rx and R2 hereinafter are defined as follows: r, = .Galfil | 2,3 6 i NeuAc cL> alBl- R2 feuAc cf-| 2,8 feuAc oC Abbreviations: Gal: NeuAc : galactose N-acetylneuraminic acid ch9oh ch9oh H2N"R2 ch2oh —°hh ^ C'rUOH c«n-r0 / H 2 ^ sodium cyanoborohydride b) Reaction of reductively aminated sugar with SPDP The derivatization was carried out in 0.1 M sodium phosphate buffer + 0.1 M sodium chloride, pH 7.5, with a 3-5-fold molar excess of SPDP. The reaction time at room temperature was 2-12 hours. ch2oh OH ch-p—nh- 0 0. ho-n; II )— S~ S-CH^- CH?~ C- 0-N — \ \ 0 / CHnOH OH \ f 0 0 It chr nh- c- ch9~ ch2~s-sh^ ; - 7 - The sugar-NH2-SPDP derivative was separated from N-hy-droxysuccinimide and unreacted SPDP on a Biogel P2 column. If the derivative was contaminated with SPDP an additional purification step was carried out on a small 5 silica gel column which was eluted with solvent mixtures of increasing polarity. c). Reaction of HSA with SPDP The protein derivatization was carried out under the same conditions as the sugar derivatization in 0.1 M sodium 10 phosphate buffer, pH 7.5, with a 3-5-fold molar excess, based on free epsilon-aminolysyl groups, of SPDP.
The protein-SPDP derivative was separated off on a Sephadex G-25 column which was eluted with the buffer for the subsequent reactions (0.1 M sodium phosphate buffer, 15 pH 6, 5 mM EDTA).
Protein -Nl^. + 0 II - 8 - d) Reduction of the HSA-SPDP derivative The disulfide bridges newly introduced by the derivatization in the HSA-SPDP derivative were reduced, with the elimination of 2-thiopyridone, in 0.1 M sodium phosphate 5 buffer, pH 6, 5 mM EDTA with the addition of 25 mM dithiothreitol. The native disulfide bridges of the protein are not reduced under these reaction conditions. The reaction was carried out at room temperature, and the reaction time was 1-2 hours. 0 II Protein—N-C-CH^- CH~-3~S H 2 2 Dithiothreitol , f "0 II Protein-N- OCH-r CH-r SH H 2 2 10 The reduced HSA-SPDP derivative was separated off on a Sephadex G-25 column using 0.1 M sodium phosphate buffer, pH 6, 5 mM EDTA as eluting buffer. e) Coupling of the sugar and of the protein derivative Reduced HSA-SPDP derivative was immediately reacted with 15 the sugar-NH2-SPDP derivative: the sugar-NH2-SPDP derivative was used in a 2-5-fold molar excess based on epsilon-aminolysyl groups in the protein, and the reaction time was 24-48 hours at room temperature. 9 - ch2oh j—oh 0 II 0 Rr\| pyN~C~CH9~ CRjS-S-^ + Protein-N-C-CH^-CH?- sh h Y // h 0 0 II CH-r N- C- CH-j-CH-fS~ S- Q-k-C'rW C- N-Prot e i n / h 2 2 h The coupling product or neoglycoprotein was separated from the other reaction products by column chromatography on Sephadex G-25.
In the procedure described, reductively aminated sugars 5 are coupled via SPDP to free epsilon-aminolysyl groups of the protein. An alternative possibility is to reduce the disulfide bridges of the protein and to couple the reductively aminated sugars via SPDP to the sulfhydryl groups, which are now free, of the cysteine residues. 10 3. Detection methods for the identification of the intermediate products The following detection methods were used by choice for the identification of the intermediate products: 15 A) for monosialolactose and disialolactose and the corresponding derivatives a) a change, after reductive amination and SPDP-derivatization, in the migration behavior on thin-layer chromatography on silica gel G-60 - 10 - plates in the following solvents: pyridine/ethyl acetate/glacial acetic acid/water (6:3:1:3) chloroform/methanol/0.2% aqueous calcium chloride (60:35:8) and stainability of the bands with iodine vapor, ninhydrin and resorcinol. b) Quantification of SPDP-derivatization by a slight modification of the determination of neuraminic acid with 1,2-diamino-4, 5-dimethoxybenzene (S. Hara et al. (1986) J. Chromatography 377, 111-119) and by elimination of 2-thiopyridone with dithiothreitol. The amount of 2-thiopyridone liberated, which corresponds to the amount of SPDP-derivatized sugar, can be measured by measuring the increase in extinction at the wavelength of lambda = 343 nm in a photometer, and the concentration can be calculated using the Lambert-Beer law. The molar extinction coefficient for 2-thiopyridone at lambda = 343 nm is 7.06 x 103 x M"1 x cm"1 (D.R. Grassetti & J.F. Murray, (1967) Arch. Biochem. Biophys. 119, 41-49) . c) GC Analysis of the individual sugar derivatives after reductive amination and SPDP-derivatization by the method of J. Lechner et al. (1985) J. Biol. Chem. 260, 860-866.
For HSA a) Protein determination was carried out with the Bio-Rad protein assay. b) the SPDP-derivatization was quantified by protein determination with the Bio-Rad protein assay and by elimination of 2-thiopyridone with dithio- - 11 - threitol. The liberated 2-thiopyridone, which corresponds to the amount of bound SPDP, was measured by the increase in extinction at the wavelength lambda = 343 nm in a photometer, and 5 the concentration was calculated using the Lambert-Beer law. The molar extinction coefficient for 2-thiopyridone at lambda = 343 nm is 7.06 x 103 x M"1 x cm"1 (Grassetti & Murray, loc. cit.). 10 c) HSA and HSA-SPDP derivatives show different migration behaviors on SDS gel electrophoresis, stained with Coomassie blue. 4. Synthesis of disialolactose-HSA conjugate In a characteristic experimental mixture, 10 mg 15 (10.75 (imol) of disialolactose in 2 ml of a methanolic ammonium acetate solution were reductively aminated with the addition of 20 mg of sodium cyanoborohydride. The reductively aminated sugar was purified on a Biogel P2 column and then, in 13 ml of 0.1 M sodium phosphate 2 0 buffer, pH 7.5, reacted with 13 mg of SPDP dissolved in 1050 (il of ethanol, and the disialolactose-NH2-SPDP derivative was purified on a Biogel P2 column and a silica gel column. The yield, measured by determination of neuraminic acid with 1,2-diamino-4, 5-dimethoxybenzene 25 and by elimination of 2-thiopyridone with dithiothreitol, was about 50 % (4.8-5.2 ^mol) of the amount of disialolactose used.
For the derivatization of HSA, 5.6 mg of SPDP were dissolved in 450 jul of ethanol and added to 6 mg of HSA 30 in 5550 /xl of 0.1 M sodium phosphate buffer, pH 7.5. Purification on a Sephadex G-25 column resulted in about 4.8 mg of HSA-SPDP derivative with a derivatization of 45-53 SPDP=molecules per HSA molecule.
The disulfide bridges newly introduced in the HSA-SPDP - 12 - derivative were cleaved with 25 mM dithiothreitol in 0.1 M sodium phosphate buffer + 5 mM EDTA, pH 6. The reaction time was 1 hour at room temperature. After purification on a Sephadex G-25 column, the reduced 5 HSA-SPDP derivative was immediately reacted with the disialolactose-NH2-SPDP derivative in 0.1 M sodium phosphate buffer + 5 mM EDTA, pH 6. It was possible to follow the coupling reaction in a photometer by an increase in the extinction at the wavelength lambda = 343 nm due to 10 the elimination of 2-thiopyridone. There was no further increase in extinction after 24-48 hours, the coupling reaction was complete.
Purification of the coupling product on a Sephadex G-25 column resulted in 2.1-2.5 mg of disialolactose-HSA 15 conjugate which was derivatized with 30-33 disialolactose molecules per HSA molecule.
The degree of derivatization of the coupling product was determined by 1.) protein determination with the Bio-Rad protein assay and 2.a) by determination of neuraminic 20 acid in the coupling product with 1,2-diamino-4,5-dimethoxybenzene, b) by 'calculating the concentration of liberated 2-thiopyridone (in the final coupling step) by measuring the extinction at the wavelength lambda = 343 nm and inserting the appropriate parameters into the 25 Lambert-Beer equation and c) by determining the concentration of recovered unreacted disialolactose-NH2-SPDP derivative using the methods mentioned under a) and b). 5. Immunological reactions of the disialolactose-HSA conjugate 3 0 The coupling product (disialolactose-HSA conjugate) was subsequently tested in a Bio-Dot assay with monoclonal anti-GD3 antibodies: both- mab 704/16 and mab 624/253 (both directed against the GD3 ganglioside) reacted with the neoglycoprotein. - 13 - It was also possible after SDS gel„electrophoresis under non-reducing conditions and subsequent Western blotting to show the reaction of the monoclonal antibodies mab 704/16 and mab 625/253 with the disialolactose-HSA conjugate.
Immunization experiments on mice have shown that the disialolactose-HSA conjugate is considerably more immunogenic than the GD3 ganglioside, i.e. elicits a stronger humoral immune response.
Claims (15)
1. A neoglycoprotein consisting of a carrier protein and the sugar mono- or disialolactose. 5
2. A neoglycoprotein as claimed in claim 1, wherein the carrier protein is human serum albumin (HSA).
3. . A process for the preparation of a neoglycoprotein as claimed in claim 1, which comprises coupling the sugar to the carrier protein.
4. 1.0 ' 4. The process for the preparation of a neoglycoprotein as claimed in claim 1, which comprises covalently coupling the sugar to the carrier protein.
5. The process for the preparation of a neoglycoprotein as claimed in claim 4, wherein N-succinimidyl 3-(2- 15 pyridyldithio)propionate (SPDP) is used as coupling reagent.
6. The process for the preparation of a neoglycoprotein as claimed in claim 3, wherein bovine colostrum is used as source of sugar. 20
7. The use of a neoglycoprotein as claimed in claim 1 or obtainable as claimed in claim 3 for the production of a vaccine.
8. The use of a neoglycoprotein as claimed in claim 1 or obtainable as claimed in claim 3 for the produc- 25 tion of a diagnostic agent.
9. The use of a neoglycoprotein as claimed in claim 1 in a competitive radioimmunoassay for detecting gangliosides.
10. A vaccine which contains a neoglycoprotein as 3 0 claimed in claim 1.
11. A neoglycoprotein as claimed in claim 1, substantially as hereinbefore described and exemplified.
12. A process for the preparation of a neoglycoprotein as claimed in claim 1, substantially as hereinbefore described and exemplified.
13. A neoglycoprotein as claimed in claim 1, whenever prepared by a process claimed in a preceding claim.
14. Use as claimed in claim 8 or 9, substantially as hereinbefore described.
15. A vaccine as claimed in claim 10, substantially as hereinbefore described. F. R. KELLY 4 CO., AGENTS FOR THE APPLICANTS.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3837623A DE3837623A1 (en) | 1988-11-05 | 1988-11-05 | NEOGLYCOPROTEINS, THEIR PRODUCTION AND USE |
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| IE893548L true IE893548L (en) | 1990-05-05 |
| IE76312B1 IE76312B1 (en) | 1997-10-22 |
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| EP (1) | EP0368131B1 (en) |
| JP (1) | JP2838147B2 (en) |
| KR (1) | KR0162635B1 (en) |
| AT (1) | ATE142229T1 (en) |
| AU (1) | AU630634B2 (en) |
| CA (1) | CA2002218C (en) |
| DE (2) | DE3837623A1 (en) |
| DK (1) | DK549589A (en) |
| ES (1) | ES2093612T3 (en) |
| GR (1) | GR3021111T3 (en) |
| IE (1) | IE76312B1 (en) |
| PT (1) | PT92187B (en) |
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| DE3840044A1 (en) * | 1988-11-27 | 1990-06-07 | Behringwerke Ag | GLYCOSPHINGOLIPIDS WITH A COUPLABLE GROUP IN THE SPHINGOID PART, THEIR PRODUCTION AND USE |
| EP0646377A1 (en) * | 1993-10-01 | 1995-04-05 | LUANFARMA S.r.l. | Albumin-conjugated tumour cell-free extracts, antigenic compositions comprising the same, related antibodies, and uses thereof |
| US5612030A (en) | 1995-01-17 | 1997-03-18 | University Of Kentucky Research Foundation | Anti-idiotype monoclonal antibody 1A7 and use for the treatment of melanoma and small cell carcinoma |
| US5935821A (en) | 1995-01-17 | 1999-08-10 | Board Of Trustees Of The University Of Kentucky | Polynucleotides related to monoclonal antibody 1A7 and use for the treatment of melanoma and small cell carcinoma |
| US5977316A (en) * | 1995-01-17 | 1999-11-02 | The Board Of Trustees Of The University Of Kentucky | Monoclonal antibody 1A7 and related polypeptides |
| AU6485496A (en) * | 1995-07-14 | 1997-02-18 | Glycotech Corp. | Compounds and methods for treatment of egf receptor associated cancers and purification of the egf receptor |
| US6355244B1 (en) | 1997-11-17 | 2002-03-12 | University Of Kentucky Research Foundation | Methods and compositions for the treatment of psoriasis |
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| DE3840044A1 (en) * | 1988-11-27 | 1990-06-07 | Behringwerke Ag | GLYCOSPHINGOLIPIDS WITH A COUPLABLE GROUP IN THE SPHINGOID PART, THEIR PRODUCTION AND USE |
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1988
- 1988-11-05 DE DE3837623A patent/DE3837623A1/en not_active Withdrawn
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1989
- 1989-11-02 EP EP89120245A patent/EP0368131B1/en not_active Expired - Lifetime
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- 1989-11-03 DK DK549589A patent/DK549589A/en not_active Application Discontinuation
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| JPH02173000A (en) | 1990-07-04 |
| PT92187A (en) | 1990-05-31 |
| CA2002218A1 (en) | 1990-05-05 |
| EP0368131B1 (en) | 1996-09-04 |
| DK549589A (en) | 1990-05-06 |
| AU4436889A (en) | 1990-05-10 |
| IE76312B1 (en) | 1997-10-22 |
| ES2093612T3 (en) | 1997-01-01 |
| KR900007429A (en) | 1990-06-01 |
| KR0162635B1 (en) | 1998-11-16 |
| ATE142229T1 (en) | 1996-09-15 |
| DE3837623A1 (en) | 1990-05-10 |
| PT92187B (en) | 1995-06-30 |
| DK549589D0 (en) | 1989-11-03 |
| EP0368131A2 (en) | 1990-05-16 |
| DE58909720D1 (en) | 1996-10-10 |
| GR3021111T3 (en) | 1996-12-31 |
| CA2002218C (en) | 2000-08-08 |
| EP0368131A3 (en) | 1990-12-19 |
| AU630634B2 (en) | 1992-11-05 |
| JP2838147B2 (en) | 1998-12-16 |
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