CA2265887A1 - Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof - Google Patents
Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof Download PDFInfo
- Publication number
- CA2265887A1 CA2265887A1 CA002265887A CA2265887A CA2265887A1 CA 2265887 A1 CA2265887 A1 CA 2265887A1 CA 002265887 A CA002265887 A CA 002265887A CA 2265887 A CA2265887 A CA 2265887A CA 2265887 A1 CA2265887 A1 CA 2265887A1
- Authority
- CA
- Canada
- Prior art keywords
- shiga
- toxin
- toxins
- toxoids
- polypeptide
- 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
- 239000003053 toxin Substances 0.000 title claims abstract description 77
- 108700012359 toxins Proteins 0.000 title claims abstract description 77
- 231100000765 toxin Toxicity 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 59
- 238000000746 purification Methods 0.000 title claims abstract description 7
- 108010017898 Shiga Toxins Proteins 0.000 title abstract description 42
- 238000002360 preparation method Methods 0.000 title description 3
- 230000012743 protein tagging Effects 0.000 title description 2
- 108010079723 Shiga Toxin Proteins 0.000 claims abstract description 62
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 16
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 16
- 238000002955 isolation Methods 0.000 claims abstract description 3
- 208000015181 infectious disease Diseases 0.000 claims description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 14
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 13
- 229960005486 vaccine Drugs 0.000 claims description 13
- 229920001184 polypeptide Polymers 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 7
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 4
- 231100000033 toxigenic Toxicity 0.000 claims description 4
- 230000001551 toxigenic effect Effects 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- FMYBFLOWKQRBST-UHFFFAOYSA-N 2-[bis(carboxymethyl)amino]acetic acid;nickel Chemical compound [Ni].OC(=O)CN(CC(O)=O)CC(O)=O FMYBFLOWKQRBST-UHFFFAOYSA-N 0.000 claims description 2
- 239000003446 ligand Substances 0.000 claims description 2
- 230000001404 mediated effect Effects 0.000 claims 1
- 244000052616 bacterial pathogen Species 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 31
- 239000013612 plasmid Substances 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 18
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 description 17
- 230000004927 fusion Effects 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- 150000001768 cations Chemical class 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 10
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 241000282412 Homo Species 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 108010090763 Shiga Toxin 2 Proteins 0.000 description 9
- 150000001413 amino acids Chemical group 0.000 description 9
- 231100000135 cytotoxicity Toxicity 0.000 description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- 230000003013 cytotoxicity Effects 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 210000003501 vero cell Anatomy 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000028993 immune response Effects 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 241000607764 Shigella dysenteriae Species 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000021615 conjugation Effects 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 238000006386 neutralization reaction Methods 0.000 description 5
- 230000001681 protective effect Effects 0.000 description 5
- 229940007046 shigella dysenteriae Drugs 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 102000011931 Nucleoproteins Human genes 0.000 description 4
- 108010061100 Nucleoproteins Proteins 0.000 description 4
- 108010039491 Ricin Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 229930186900 holotoxin Natural products 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 206010014096 Echinococciasis Diseases 0.000 description 3
- 208000009366 Echinococcosis Diseases 0.000 description 3
- 206010014896 Enterocolitis haemorrhagic Diseases 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 235000015220 hamburgers Nutrition 0.000 description 3
- 238000011031 large-scale manufacturing process Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- HFQKBOPMAOTAIR-TZSVBWBLSA-N α-d-galactosyl-(1->4)-β-d-galactosyl-(1->4)-β-d-glucosylceramide Chemical class O[C@@H]1[C@@H](O)[C@H](OC[C@@H]([C@H](O)/C=C/CCCCCCCCCCCCC)NC(C)=O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](CO)O1 HFQKBOPMAOTAIR-TZSVBWBLSA-N 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 244000153158 Ammi visnaga Species 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101710173438 Late L2 mu core protein Proteins 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 208000031513 cyst Diseases 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 229940023146 nucleic acid vaccine Drugs 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 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
- 238000000527 sonication Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 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
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- WIGDGIGALMYEBW-LLINQDLYSA-N 2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]propanoyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]acetic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O WIGDGIGALMYEBW-LLINQDLYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 208000003508 Botulism Diseases 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 206010012741 Diarrhoea haemorrhagic Diseases 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 108091006054 His-tagged proteins Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108090000829 Ribosome Inactivating Proteins Proteins 0.000 description 1
- 101150054830 S100A6 gene Proteins 0.000 description 1
- 101100391171 Schizosaccharomyces pombe (strain 972 / ATCC 24843) for3 gene Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229940044197 ammonium sulfate Drugs 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 235000021168 barbecue Nutrition 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002242 enterotoxic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 235000013410 fast food Nutrition 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 230000000521 hyperimmunizing effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 229960000318 kanamycin Drugs 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
- 229930182823 kanamycin A Natural products 0.000 description 1
- 108010082683 kemptide Proteins 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 230000001459 mortal effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 231100000189 neurotoxic Toxicity 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- -1 peptides Chemical class 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 206010040400 serum sickness Diseases 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 239000012138 yeast extract Substances 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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- 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
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/25—Shigella (G)
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention describes the isolation and purification of biologically and immunologically active histidine-tagged Shiga toxins (His-tagged), a toxin associated with HC and the potentially life-threatening sequela HUS transmitted by strains of pathogenic bacteria. The present invention describes how his-tagging greatly simplifies and expedites purifying Shiga toxins, and describes an improved method for such purification. One aspect of the invention is obtaining and using Shiga toxoids that are immunoreactive but not toxic. Another aspect of the invention is obtaining and using fusion proteins of His-tagged Shiga toxins or toxoids. Yet another aspect of the invention is obtaining and using antibodies to His-gagged Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins.
Description
15202530W0 98/ 1 1229CA 02265887 1999-03-09PCT/US97I15836-1-DescriptionHISTIDINE-TAGGED SHIGA TOXINS, TOXOIDS, ANDPROTEIN FUSIONS WITH SUCH TOXINS AND TOXOIDS,METHODS FOR THE PURIFICATION AND PREPARATION THEREOF§iQVERNME[\_lT INTERESTThe invention described herein may be manufactured, licensed, and used forgovernmental purposes without payment of royalties to us thereon.FIELD OF THE INVENTIONThe invention relates to a family of multi-unit bacterial proteins that areassociated with hemorrhagic colitis and the lifeâthreatening sequela, hemolytic uremicsyndrome. These proteins, defined as members of the âSl1iga toxin family," have beentagged with histidine residues. The invention further relates to a non-toxinogenic butimmunoreactive form of histidineâtagged Shiga toxins, or toxoids. Moreover, theinvention relates to fusion proteins obtained by combining histidineâtagged Shigatoxins or toxoids with other proteins. Histidine tagging greatly facilitates puriï¬cationof Shiga toxins, and the invention also relates to methods for purifying such toxins.The invention further relates to using the histidineâtagged Shiga toxoids or fusionproteins of Shiga toxoids as antigens for generating an immune response againstinfection or transmission by bacteria expressing Shiga toxin. It also relates toantibodies to Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins, bothmonoclonal and polyclonal, and their use in treating, diagnosing, and preventing ofdisease and infections by pathogenic E. coli. Finally, the invention relates to preparingthe Shiga toxins, toxoids, and fusion proteins.BACKGROUND OF THE INVEN I IONEnterohemorrhagic Escherichia coli (EHEC) are associated with food-bomeoutbreaks of bloody diarrhea or "hemorrhagic colitis" (HC) and the hemolytic uremicsyndrome (HUS). (Spika, J. et al., "Hemolytic Uremic Syndrome and DiarrheaAssociated with Escheria coli: 01 57:H7 in a Day Care Center," J. Pediatr., 109: 287-291 (1 986); Remis, R., "Sporadic case of hemorrhagic colitis associated with Escheriacoli 0157:H7," Ann. Intern. Med, 101 :624-626 (1984); "Riley, L. et al., "Hemorrhagic10152025W0 98/ 1 1229CA 02265887 1999-03-09PCT/US97I15836-2-colitis associated with a rare Escheria cali serotype," N. Engl. J Med., 308:681-685(1983)). EHEC infection can be deadly and poses a signiï¬cant threat to the young andthe elderly, who are the most likely to develop serious complications from EHECinfections. Several outbreaks and sporadic cases of HC and HUS have occurred overthe past few years, with the largest outbreak in United States in 1993. In that outbreak,over 500 cases of HC and HUS were traced to contaminated hamburgers from a Jack-in-the Box fast food restaurant. (Centers for Disease Control and Prevention, Morbid.Mortal. Weekly Rep., 42:258( 1993)). In July 1996, a large outbreak of EHEC in Japanresulted in over 10,000 infected individuals and 8 deaths. Many Japanese childrenrequired hospitalization. Unfortunately, no cure or vaccine for HC and HUS iscurrently available.Primarily, HC and HUS are transmitted by the ingestion of contaminated food,particularly undercooked beef products, such as hamburger. (Doyle et al., Appl.Environ. Microbial. 53:2394 (1987); Samadpour et al., J. Appl. Environ. Microbial.60:l038 (1994)). With the prevalence of EHEC in cattle and the subjective nature ofdifferentiating between cooked and undercooked hamburgers, a stop at a fast foodrestaurant or a family barbecue can result in tragedy. HC and HUS appear to bemediated by the toxin produced by EHEC and Shigella dysenteriae (for review seeO'Brien and Holmes, Microbial. Rev., 51: 206-220 (1987)). These bacteria producea family of closely related cytotoxins that collectively will be called "Shiga toxins" forthe purpose of this application. Shiga toxins (alternatively, âverotoxins") havecytotoxic, neurotoxic, and enterotoxic activity (Strockbine, N. et al., "Two toxin-converting phages from Escheria cali 01 57:H7 strain 933 encode antigenically distincttoxins with similar biological activities," Infect. Immun., 53:135-140 (1986)).Based on their immunological cross-reactivity, the Shiga toxins have beendivided into two groups. (Strockbine et al., supra). These groups have beendesignated Shiga toxin type 1 (Stxl) and Shiga toxin type 2 (Stx2). (Strockbine et al.,supra; Calderwood et al., "Proposed New Nomenclature for SLT (VT) Family," ASMNews, 62:l18â1l9 (1996)). The Stxl group contains the prototype Stxl toxin from10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97I 15836-3-EHEC as well as the Shiga toxin from Shigella dysenteriae type 1. In recent years,other types of toxins have been discovered and considered members of the Stx2 group.These are Stx2e, Stx2c, Stx2vha, and Stx2vhb. (Lindgren et al., Infection andImmunology, 6113832 (1993); Schmitt, C. et al., "Two copies of Shiga-like toxin II-related genes common in enterohemorrhagic Escheria coli strains are responsible forthe antigenic heterogeneity of the 0157:H strain E32511," Infect. Immun., 59: 1065-1073(1991); Marques, L. et al., "Escheria coli strains isolated from pigs with edema diseaseproduce a variant of Shiga-like toxin Il," FEMS Lett., 44:33-38 (1987)).For the purposes of this application the term "Shiga toxin" encompasses Shigatoxin and any other toxins in the Stxl or Stx2 group. The abbreviation "Stx" will referto the protein designation, and the abbreviation "Stx" to the gene designation.These Shiga toxins do share similar genetic and protein organization, as setforth in Figure l. The A subunit gene encodes the enzymatically active subunit. TheA subunit polypeptide has two functional domains, Al and A2, which are linked bya disulfide bond. The Al portion is an N-glycosidase that acts on the 28S rRNAsubunit of eukaryotic ribosomes to inhibit protein synthesis. (Saxena, S. et al., "Shigatoxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of28S RNA when microinjected into Xenopus ooeytes," J. Biol. Chem., 264:596-601(1989)). The A2 fragment is required for the binding of 5 B subunit polypeptides. Thepentamer of B subunits is responsible for binding to a receptor on eukaryotic cells. Apolypeptide containing the entire A subunit and B subunit pentameter is referred to asa Shiga holotoxin. Despite this knowledge about the toxin components, there is noknown cure or vaccine for HC or HUS.The need exists for therapeutic agents for the treatment and prevention of HCand HUS. However, progress in the search for such agents has been hampered by thelack of a fast and simple method for purifying Shiga toxins. Therefore, the need existsfor such a fast and simple method. Moreover, the need exists for such a method thatfurther allows for large-scale production of Shiga toxins while retaining theirbiological and immunological activity. The need also exists for such a method that10152025W0 98/ 11229CA 02265887 1999-03-09PCT/US97/15836-4-allows for large-scale production of Shiga toxoids and fusion proteins of Shiga toxinsand toxoids. Such a method should simplify obtaining antibodies against Shiga toxinand vaccines against HC and HUS using Shiga toxoids and fusions of Shiga toxoids.SUMMARY OF THE INVENTIONThe present invention describes the isolation and puriï¬cation of biologicallyand immunologically active histidineâtagged Shiga toxins (His-tagged), a toxinassociated with HC and the potentially life-threatening disease HUS transmitted bystrains of pathogenic bacteria. The present invention describes how his-tagging greatlysimplifies and expedites purifying Shiga toxins, and describes an improved method forsuch puriï¬cation.One aspect of the invention is obtaining and using Shiga toxoids that areimmunoreactive but not toxic. For example, the invention describes using suchobtained Shiga toxoids in vaccines against HC and HUS.Another aspect of the invention is obtaining and using fusion proteins of His-tagged Shiga toxins or toxoids. These fusion proteins have the advantage ofcombining beneï¬cial properties of each protein, resulting, for example, in improvedprotein stability or targeted delivery of a his-tagged Shiga therapeutic agent.Yet another aspect of the invention is obtaining and using antibodies to His-tagged Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins. These antibodiescan be either monoclonal or polyclonal and have potential uses in treating, diagnosing,or preventing HC and HUS caused by EHEC or Shigella dysenteriae type 1 infections.Other aspects of the present invention will become apparent from the moredetailed description provided below, to be read in conjunction with the accompanyingdrawings.BRIEF DESQRIPTION OF THE DRAWINGSFigure 1 depicts the protein structure of Shiga toxin genes.Figure 2 depicts the predicted amino acid sequence for the mature A subunitand the unprocessed B subunit of Stxl. (Calderwood et al., Proc. Natl. Acad. Sci.USA, 84: 4364-4368 (1987); DeGrandis et al., J. Bacterial, 169:43l3-4319(l987)).10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/ 15836-5-Figure 3 depicts the predicted amino acid sequence for the mature A subunitand the unprocessed B subunit of Stx2. (Jackson et al., FEMS Lett., 44:109-114(1987)).Figure 4 depicts the predicted DNA sequence for stxl and DNA upstream ofthat sequence. (Calderwood et al., Proc. Natl. Acad. Sci. USA, 84: 4364-4368 (1987);DeGrandis et al., J. Bacteriol., 169: 4313-4319 (1987)).Figure 5 depicts the predicted DNA sequence for stx2 and DNA upstream ofthat sequence. (Jackson et al._. FEMS Lett., 44: 109-114 (1987)).Figure 6 depicts the approximately 1200 base pair fragments of stx1 producedby PCR amplification. Figures 6aâc depict the fragments used to make plasmidspQHl, pQHEI, and p7HI, respectively. Nucleotides in lower case represent non-toxinsequences in the primers and/or base changes.Figure 7 depicts the approximately 1200 base pair fragments of stx2 producedby PCR ampliï¬cation. Figures 7a and 7b depict the fragments used to make plasmidspQHI and pQHEIl, respectively. Nucleotides in lower case represent non-toxinsequences in the primers and/or base changes.Figure 8 depicts the plasmid pQHI, encoding the His-Stx 1 fusion and drivenby the T5 promoter.Figure 9 depicts the plasmid pQHlI, encoding the His-Stx 2 fusion and drivenby the T5 promoter.Figure 10 depicts the plasmid pQHEI, encoding the His-Enterokinase site-Stxlfusion and driven by the T5 promoter.Figure 11 depicts the plasmid pQHEII, encoding the His-Enterokinase site-Stx2 fusion and driven by the T5 promoter.Figure 12 depicts the plasmid pQHIIvhb, encoding the His-Stx 2 fusion anddriven by the T5 promoter.Figure 13 depicts the plasmid pQHEllVhb, encoding the His-Enterokinase site-Stx 2 fusion and driven by the T5 promoter.10152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97/ 15836-6-Figure 14 depicts the plasmid p7HI, encoding the His-Stx 1 fusion and drivenby the PT7 promoter.Figure 15 depicts the plasmid p7HII, encoding the His-Stx 2 fusion and drivenby the PT7 promoter.Figure 16 depicts the expression of His-Stx fusion proteins according to theinvention.DETAILED DESCRIPTIQN OF THE INVENTIONAn object of the invention is to purify large quantities of Shiga toxins thatretain their biological and immunological properties. To achieve this object, a Shigatoxin gene was cloned into a Histidine-tag expression vector, expressed, and purified.An additional object of the invention is to obtain antigens speciï¬c to Shiga toxoids,a toxin that is nonâtoxinogenic but immunoreactive, for generating an immuneresponse against Shiga toxins. Another object of the invention is the creation ofantibodies against Shiga toxins or toxoids for treating, diagnosing, or preventingdisease and infections by pathogenic bacteria. The his-tagged Shiga toxins or toxoidsdescribed above can be used for these purposes.Those skilled in the art will also recognize that the size of the his-tagged Shigatoxin to be used may be varied according to the speciï¬c purpose for the Shiga toxin.For example, if the purpose is fusing the his-tagged Shiga toxin or toxoid with one ormore proteins, a smaller fragment might be selected to enhance stability of thecombined fusion product, although using a larger fragment is by no means precluded.The desired size of the His-Shiga toxin may also vary with the convenience of theavailable restriction sites, in light of the materials and methods known to those skilledin the art. Consequently, the terms "HisâShiga toxin" or "His-tagged Shiga Toxin"refers to the fragment of about 372-377 amino acids comprising the A and B subunitsof any of the Shiga toxin family members fused with a histidine tag. Smallerfragments that retain biological and/or immunological function are also included.Biological ï¬mction is measured by, for example, cytotoxicity to Vero cells, asdescribed in Example III.A. Immunological function may also be tested by, for10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/ 15836-7-example, neutralization by speciï¬c antisera, as described in Example lII.B. Apreferred embodiment of the invention is a His-Tagged Shiga holotoxin, containing1 A subunit and 5 B subunits. In another preferred embodiment, the tag consists of sixhistidine residues. The most preferred embodiment is a Hisâ,-Tagged Shiga holotoxin.One of the objects of the present invention is to administer His-Shiga toxoidsto protect against illness or disease caused by EHEC or Shigella dysenteria type I, suchas HC and HUS. The object is achieved through the stimulation of immune responsedirected against Shiga toxins.Consequently, the term "immunizing" or "immunization" is used in theapplication. The degree of protection achieved by such immunization will vary withthe degree of homology between Shiga toxins and the His-Shiga toxoids, as well asother factors, such as unique attributes of the patient or the species treated. Moreover,immunization is not limited to avoiding infection altogether; it also includesdecreasing the severity of the infection, as measured by the following indicators:reduced incidence of death, HUS, or permanent kidney damage; decreased levels oftoxin; reduced ï¬uid loss; or other indicators of illness regularly used by those skilledin the relevant art.Unless specified otherwise, the uses and methods set forth herein are generallyapplicable to humans and animals. The term "patient" is used herein to mean bothhumans and animals, and "animals" is not limited to domesticated animals but alsomay include wildlife and laboratory animals.Moreover, because His.-Tagged Shiga toxins according to the invention havethe biological and immunological properties of Shiga toxins, they may be used for anyapplication appropriate for Shiga toxins. For example, it has been recentlydemonstrated that Stxl can be used to treat bone marrow cells from mice with humanB-cell lymphomas. The Shiga toxin bound to the receptor on the lymphoma cell andthe toxin killed the cancer cell. (LaCasse et al., Blood 88:1551(l996)). Thus, theskilled artisan would expect that His-Shiga toxins or fusions could be used for thesame purpose and in the same manner.10152025W0 98/11229CA 02265887 1999-03-09PCT/U S971 15836-3-Isolating and Purifying His-Tagged Shiga ToxinThe standard protocol for puriï¬cation of Shiga toxin comprising A and Bsubunits uses biochemical techniques. The standard protocol was developed byO'Brien et al. (O'Brien et al., Infect. Immun. 402675 (1983); O'Brien et al., Infect.Immun., 30:l70(l 980)). The method employs four puriï¬cation steps: 1) ammoniumsulfate 2) DEAE Sepharoseprecipitation; column chromotography; 3)chromatofocusing; and 4) antibody affinity chromotography. This method has theadvantages of employing publicly available materials, being capable of purifying allShiga toxins, and being capable of purifying Shiga toxins for human use. Itsdisadvantage is that the minimum time required for this test is three weeks.Another well-known method for purifying Shiga toxin from bacteria wasdeveloped by Keusch et al. (Donohue-Rolfe et al., Infect. Immun. 5723888 (1989);Acheson et al., Microb. Pathog. 14:57 (1993)). This method employs a Shiga toxinreceptor analog. The receptor analog is the Pl glycoprotein (Plgp) from tapewormhydatid cysts material (HCM) in sheep gut. This method contains three puriï¬cationsteps: 1) ammonium sulfate precipitation; 2) Blue sepharose chromotography; and 3)Plgp column chromatography. The Plgp must be prepared from the HCM. Thoughfaster than the standard method, this method still requires a minimum of two or moreweeks. The hydatid cyst material must be obtained from infected sheep and is notpublicly available. The method has the additional disadvantages of being capable ofuse with only those Shiga toxins that bind Plgp and, because of possiblecontamination, it is not appropriate for obtaining Shiga toxoids for use in humans.Using recombinant methods, the Shiga toxin gene or portions of the Shigatoxin gene have been cloned and expressed in bacteria and puriï¬ed. Zollman et al.,Prot. Expression Pur. 5:291 (1994), puriï¬ed a recombinant Stxl A1 fragment.Acheson et al., Infect. Immunol. 63:301(1995), expressed and puriï¬ed the Stx2 Bsubunit. Downes et al., Infect. Immun. 5621929 (1988), expressed the stx2 gene inbacteria and puriï¬ed Stx2. However, the puriï¬cation methods following expression10152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97l 15836-9-were essentially those of the standard method or hydatid cyst method and, therefore,had the same disadvantages.In the search for a method for purifying Shiga toxins, applicants havedeveloped a purification method based on the creation of a histidineâtagged Shigatoxin. Methods for histidine tagging are known in the art. For example, Fryxell et al.,Biochem. Biophys. Res. Comm., 2102253-259 (1995), added a kemptide and histidinetag to the A chain of the eukaryotic toxin ricin, which was later associated with the Bsubunit. The ricin toxin differs from Shiga toxins in origin (prokaryotic v. eukaryotic)and structure of the B subunit (the ricin B subunit is a single polypeptide, not apentamer). Moreover, Fryxell et al. only expressed the A subunit with a His-Tag. Inaddition, Strauss et al., FEMS Microbiol. Lett., 1272249-254 (1995), have his-taggedthe C-terminus of the cholera toxin B subunit, and expressed a his-tagged B subunit-IgA protease fusion protein. However, this did not involve expressing the entire toxinwith a His-Tag, and the expressed fusion protein did not undergo multimerization.Finally, Terbush & Novick, .1 Cell. Biol., 130:299â312 (1995), tagged the C-terminusof a multiunit yeast protein. This involves a eukaryotic rather than a prokaryoticsystem. Moreover, expressing a functionally active Shiga toxin requires retaining itsmultimer conformation, as well as its receptor binding and enzymatic activity.Although His-Tagging of proteins is known, it was not expected that His-Tagging of a Shiga toxin would be successful. The skilled artisan would have believedthat a His-Shiga toxin fusion would have lost cytotoxicity, because the skilled artisanwould have expected that the attachment of a His-Tag to the amino acid terminus ofa toxin would destroy its activity. Moreover, the multi-unit toxin would have beenexpected to be more susceptible to losing toxicity upon fusion with additional aminoacids, since it is known that the toxin must retain its conformation for enzymaticactivity and for binding of the B subunits to cell receptors, and the addition of aminoacids would have been expected to destroy proper conformation. The skilled artisanwould be aware that confonnation and the charge of the molecule is critical to Shigatoxins. For example, the skilled artisan would know that deleting a few N-terminall0152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97/ 15836-10-amino acids from Stx2A destroyed enzymatic activity, as reported in Perera et al.,Infect. Immun0l., 59:829-835 (1991)). Similarly, altering the C-terminus of the Bsubunit affected toxicity. (Perera et al., supra). Additionally, Perera et al. suggestedthat the charge of the molecule plays an important role. The importance of preservingShiga toxin conformation is further underscored by ï¬ndings that the highlyhomologous StxlA and Stx2B subunits cannot be combined to form an active toxin.(Weinstein et al., Irï¬zct. Immun, 57:3743-3750 (1989)). Based on this knowledge, theskilled artisan would have expected that tagging a Shiga holotoxin with histidineresidues would have unfavorably affected conformation and charge of the toxinproduct.Surprisingly, his-tagging of Shiga toxin comprising A and B subunits generateda functional Shiga toxin, which has similar speciï¬c activity to Shiga toxin puriï¬ed bystandard methods. Moreover, the His-Shiga toxins are neutralized by monoclonalantibodies speciï¬c for Shiga toxins. Example 1 describes how to create the His-Shigatoxin fusion protein.The following examples are intended to illustrate the invention but not to limitit. The skilled artisan will understand from these examples that modiï¬cations can bemade that are still within the scope of the invention. The scope of the invention isdeï¬ned by the claims.Example IA. Construction of Plasmid Encoding His-Tagged Shiga ToxinThe HisâStx fusion clones were generated by PCR amplification of six operons,restriction enzyme digestion of the PCR products, and ligation of the fragments in-frame into the appropriate vectors. The expression vectors and primers were used toplace histidine residues at the amino acid terminus of the toxins and place theconstructs under the control of either an IPTGâinducible promoter (pQE vectors)(Qiagen, Inc., 9600 DeSoto Avenue, Chatsworth, CA 91311, 1-800-362-7737) or a T7promoter Q3t7-7) (Tabor et al., Proc. Natl. Acad. Sci. 82: 1 074 (1985)). The methodsfor obtaining HisâStx fusion clones are described in more detail below.CA 02265887 1999-03-09W0 98/11229 PCT/US97l15836-11-1. Bacterial strains and plasmids. The bacterial strains and plasmidsused in this study are shown in Table 1.Table 1. Bacterial strains and plasmids used in this study.5 Strain or plasmid Characteristic(s) Source orreferenceE. coli strainsDH5oc Host strain for cloning BRLXL1-Blue Host strain for cloning; lacl; Tcâ StratageneM15 Host strain for protein puriï¬cation Qiagen10PlasmidspJES120 Encodes stx II toxin operon apJN 25 Encodes stx I toxin operon bpSQ543 Encodes stx IIvhb operon c15 pQE30 Histidine fusion vector QiagenpQE32 Histidine fusion vector QiagenpREP4 lacl; Knâ QiagenpT7-7 T7 expression vector dpGP1-2 Encodes T7 RNA polymerase; KNâ d20 a = Lindgren et al., Infect. Immunol. 61 :3832 (1993).b= Newland et al., "Cloning of shiga-like toxin structural genes from a phage ofEscheria coli strain933, in Advances in Research on Cholera and Diarheas (S. Kuwahara & N.F.Pierce eds. 1994).25 c= Lindgren et al., Infect. Immunol. 62:623 (1994).d= Tabor et al., Proc. Natl. Acad. Sci. USA 8221074 (1985).2. Media and enzymes. Bacterial strains were grown in L broth (perliter: 10 g tryptone, 5 g yeast extract, 5 g NaCl). Kanamycin, tetracycline, and30 ampicillin (Sigma Chemical Co., St. Louis, M0.) were added to the medium at ï¬nalconcentrations of 25, 10, and 100 ug/ml (respectively) as needed. RestrictionCA 02265887 1999-03-09WO 98111229 PCT/US97Il5836-12-endonucleases, calf intestinal phosphatase, and ligase were from BoehringerMannheim, Indianapolis, Ind., or U.S. Biochemicals Corporation, Cleveland, Ohio.Enzymes were used according to manufacturerâs instructions.3. Primers and PCR. The Shiga toxin genes are cloned using polymerase5 chain reaction (PCR), a standard technique in the art. Primers were designed toamplify the stx toxin operons beginning at the ï¬rst codon of the mature A subunit geneand ending downstream of the termination codon of the B subunit gene and createdusing standard techniques. The primers contained recognition sequences to generateunique restriction sites at the ends of the toxin operon. In a preferred embodiment, the10 5' primers also contained sequences to encode the recognition sequence of the proteaseenterokinase to allow for removal of the histidine residues. The primers used areshown in Table 2.TABLE 2. Primers used.15 Primer Primer Sequent (5'-3') Restrictionsitemac GCGGATCCGATGACGATGACAAACGGGAGTTTACGATAGACTT BamHlIIBAM GCGGATCCGGGAGTTTACGATAGACTT BamHlIIH3 CCACGAATAAGCTTATGCCTCA HindIII20 IBAM5 GCGGATCCAAGGAATTTACCTTAGACTTC BamHlIEC5 GCGGATCCGATGACGATGACAAAAAGGAATTTACCTTAGACTTC BamHlIPST3 ATTTTCACTGCAGCTATTCTG PstlSLTIIH5 GCATATGCATCACCATCACCATCACCGGGAGTTTACGATAGAC NdelSLTIH5 GCATATGCATCACCATCACCATCACAAGGAATTTACCTTAGECTTC Ndel25 SLTLIC3 TAACATTTATCGATATCTCCGCCTG ClalSequences encoding stx toxins were ampliï¬ed from toxin clones using a PCRkit (GeneAmp kit, Perkin-Elmer Cetus, Norwalk, CT), which was used according tothe manufacturer's instructions. The resulting stx PCR products were approximately1015202530W0 98/1 1229CA 02265887 1999-03-09PCT/US97l 15836-13-1200 bp are shown in Figures 6a, 6b, 6c, 7a, and 7b. The DNA products contain thecoding sequences for the mature A subunit and the unprocessed B subunit.Procedures for cloning are well known in the art and are described in Maniatis,Molecular Cloning: A Laboratory Manual (1982)).4. DNA manipulations. Plasmid DNA was isolated by the method ofHolmes and Quigley, Anal. Biochem., 1142193-197 (1981). Alternatively, plasmidDNA was purified using Qiagen columns (Qiagen Inc., Chatsworth, CA). PCRproducts were digested with restriction endonucleases and ligated into the pQE30/32vectors (Qiagen, Inc.), or into the vector pT7-7. PCR reactions and ligations aresummarized in Table 3.TABLE 3: PCR Reactions and ligations.Plasmid Primer Resulting clonetemplate pair Vector Cloning sitespJES 120 IIEC + IIH3 pQE30 BamHI/Hindlll pQHEIlpJESl2O IIBAM + IIH3 pQE32 BamHl/Hindlll pQHIlpJN25 IECS + IPST3 pQE30 BamHI/Pstl pQHEIpJN25 IBAM + lPST3 pQE30 BamHI/Pstl pQHIpJESl2O SLTIIH5 + IIH3 pT7-7 Ndel/Hindlll p7HIIâpJN25 SLTIH5 + SLTIC3 pT7-7 Ndel/Clal p7HlpSQ543 IIEC + IIH3 pQE30 BamHl/Hindlll pQHEIIvhbâpSQ543 [IBAM + IIH3 pQE32 BamHI/Hindlll pQHlIvhbââ The construction of these plasmids is in progress.To illustrate, the clone pQHEII was constructed as follows:Plasmid pJESl2O was the template with primers IIEC and IIH3 in a PCRreaction for ampliï¬cation of the stx2 operon. The resulting PCR product started withthe ï¬rst codon of the mature A subunit gene, extended through the A subunit gene, thecomplete B subunit gene, and ended just downstream of the terminate codon of the Bsubunit gene (Figure 6b). The PCR product was digested with the restrictionendonucleases Bam HI and Hind III, as was the vector plasmid pQE30. The vector10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-14-pQE30 was chosen because ligation of the PCR product with pQE30 at the BamHIsites would result in an in-frame protein fusion of the 6 histidine residues, theenterokinase cleavage site, additional amino acids, and the +1 residue of the matureA subunit. The digested PCR product was ligated into the digested vector pQE30.The ligation reaction was transformed into strain XL1-Blue and plated on agar thatcontained ampicillin. Colonies were screened for the presence of a plasmid thatcontained an approximately 1200 bp BamHI/HindIII DNA insert. Clones wereconï¬rmed by IPTG induction of toxin expression (Example II) with a subsequent testfor cytoxicity on vero cells (Example III). Positive clones were then transformed intoMl 5(pREP4) for large scale production of toxin.Example IILarge scale purification of His-Tagged Shiga ToxinsHis-Shiga toxin was puriï¬ed under nondenaturing conditionsibecause of themulti-subunit nature of the Shiga toxins. The strain was streaked onto a selective agarplate and incubated at 37°C for 18-24 hrs. A 20 ml overnight culture was thenprepared from a colony. The saturated culture was then diluted 1/50 into one liter ofL broth with antibiotics and the culture was grown at 37°C until it reached an O.D.600of 0.7-0.9. IPTG (2mM ï¬nal concentration) was then added to induce expression ofthe His; tagged toxin and the culture was grown for an additional 5 hrs. Cells werepelleted and the pellet was kept at -70°C overnight. The pellet was resuspended insonication buffer (50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, 20mM imidazole, 30 ug/ml PMSF), and the cells were sonicated to release toxin.Alternatively, the cells were treated with polymixin-B (2 mg/ml ï¬nal concentration)for 3 hrs at 4°C. The extracts were clariï¬ed by centrifugation and ï¬ltered through amilipore 0.45 pm ï¬lter.The nickel-nitrilotriacetic acid ligand (Ni-NTA) gel was equilibrated withsonication buffer and the cell extract was added to the gel. Protein was allowed to bindfor 1 hr at room temperature or at 4°C. The gel was washed with sonication bufferfollowed by wash buffer (50 mM sodium phosphate (pH 8.0), 300 mM sodium1015202530W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-15-chloride, 20 mM imidazole, 30 ug/ml PMSF, 10% glycerol, 1% tweenâ20). Proteinwas eluted from the gel with a gradient of imidazole (0-500 mM in wash bufferwithout tween-20), and 1 ml fractions were collected.Fractions were tested for cytotoxicity on Vero cells (as explained in ExampleIII) and were subjected to SDS-PAGE and silver stain. Fractions that were highlycytotoxic and relatively clean were pooled and dialyzed against sonication buffer. Thispool was then placed onto a Ni-NTA spin column (Qiagen) to further purify the His6-toxin and the resulting two fractions were dialyzed against PBS. A ï¬nal cytotoxicityassay and BCA protein assay were performed for the determination of the speciï¬cactivity of the puriï¬ed toxin.The protocol described above is modiï¬cation of the non-denaturing protocoldescribed by Qiagen to purify His-tagged proteins. However, the toxin that elutedcontained many contaminants. To achieve purer His-Shiga toxin, modiï¬cations weremade. Speciï¬cally, Tween-20 was added to the wash buffer, and the pH of the washbuffer was adjusted to 8. Also, a ï¬nal Ni-NTA spin column was added.This one-step His-afï¬nity method for purifying His-Shiga toxin by an Ni-NTAcolumn has several advantages over existing methods, as summarized in Table 4.TABLE 4. Comparison of Toxin Puriï¬cation TechniquesPuriï¬cation Minimum time Stepsâ Materials available Use for allMethod requiredâ Shiga toxinsStandard 3 weeks 4 yes yesHydatid 2 weeks + 3 no noCystHisé afï¬nity 1 week 2 yes yes3 Time from streaking the strain onto an agar plate. This does not include thepreparation of Plgp from hydatid cyst material which takes a minimum of 1.5 weeks.b This does not include the multiple steps involved in the puriï¬cation of Plgp fromhydatid cyst material and preparation of the column.The Ni-NTA one-step method is superior because of its relative speed andsimplicity. It requires a minimum of one week as opposed to a minimum of two or10152025W0 98/ 11229CA 02265887 1999-03-09PCTIUS97/15836-16-more weeks. Moreover, all of the materials are readily available, the method is notlimited to Shiga toxins that bind Pl gp, and the products are suitable for use in humans.The Shiga toxin obtained by the method has many uses. For example, the His-Shiga toxin may be used as a positive control antigen in a Shiga toxin detection kit.Such kits will use a puriï¬ed His-Shiga toxin as positive indicator for the toxin in asample. Other uses are detailed in the Examples below.Example IIIVerifying Biological and Immunological Activity of His-Shiga ToxinsA. Vero Cell Cytotoxicity AssayThe cytotoxicity of His-Shiga toxins obtained according to the methodsdescribed in Examples 1 and II was veriï¬ed by determining their cytotoxicity for Verocells. Cytotoxicity assays on strains that expressed His-Shiga toxins were doneessentially as described by Gentry and Dalrymple, J Clin. Microbiol, 12: 361-366(1980). Brieï¬y, cultures induced for the expression of His-Shiga toxins were disruptedby sonic lysis and clariï¬ed by centrifugation. The extracts were serially diluted intissue culture medium (Dulbecco modiï¬ed Eagle medium containing 10% fetal calfserum, 0.8 mM glutamine, 500 U of penicillin G per ml, and 500 mg of streptomycinper ml). One hundred microliters of 10-fold dilutions of the lysates were added tomicrotiter plate wells containing about 104 Vero cells in 100 pl of medium. The tissueculture cells were incubated at 37°C in 5% CO2 for 48 hours and then ï¬xed and stainedwith crystal violet. The intensity of color of the ï¬xed and stained cells was measuredwith a Titertek reader at 620 nm.B. Antisera Neutralization AssayHis-Shiga toxins obtained according to the methods described in Examples Iand II were tested for antisera neutralization. Neutralization of cytoxic activity wasdescribed in great detail in Schmitt et al., Infect. and Immun., 59:1065-1073 (1991).Brieï¬y, lysates were incubated with serial dilutions of monoclonal or polyclonalantisera speciï¬c for Stxl or Stx2 at 37°C for 2 hours. One hundred microliters of the10152025W0 98/11229CA 02265887 1999-03-09PCT/US97I15836_17_samples were then added to vero cells as described above. Percent neutralization wasdetermined by the following formula:{ [A520(toxin + antibody) - A620(toxin)]/A 620(untreated cells)} x 100.Example IVConstructing Fusions with His-Shiga Toxins and Other ProteinsUsing methods well-known in the art, the His-Shiga toxin could be fused withanother protein of interest. These methods include chemical and genetic methods, asin cloning and expressing a fusion protein, although one skilled other methods arereadily apparent to one skilled in the art. (D.V. Goeddell, Meth. Enzymol. Vol.l85(1990); Itakura, Science 19821056 (1977)). For example, if a combination vaccinefor immunization against Shiga toxin and another toxin (protein X) is desired, thenthese two toxins can be fused into a single protein. This can be achieved by firstcloning the codons for the histidine residues in frame to the coding region of proteinX. The fragment containing His-Protein X is then subcloned in-frame of the Shigatoxin operon. In a preferred embodiment, the fragment is subcloned in-frame to theA2-B portions of the Shiga toxin operon. The resulting His-Protein X-A2-B5 fusionwould ideally result in immunization against Shiga toxin and protein X.One skilled in the art would recognize that various proteins from pathogens andhaptens may be conjugated to a His-Shiga toxin. Haptens and antigens may derivefrom but are not limited to bacteria, rickettsiae, fungi, viruses, parasites, drugs, orchemicals. They may include, for example, small molecules such as peptides,oligosaccarides, and toxins. Certain antimicrobial drugs, chemotherapeutic drugshaving the capacity of being absorbed into the intestine may also be coupled to Shigatoxin for targeted delivery, since the B subunit pentamer binds to receptors in theintestine. Conjugation methods are well known in the art. Exemplary methods are setforth in Goeddel, "Systems for Heterologous Gene Expression," Meth. Ezymol., 185(1990), Itakura, "Expression in E. callâ of a chemically synthesized gene for thehormone somatostatin," Science, 198:l056âl063 (1977), and Goeddel et a1.,10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-13-"Expression of chemically synthesized genes for human insulin," Proc. Natl. Acad.Sci. USA, 281: 544-548(l979).Conjugation may be achieved by genetically fusing His-Shiga toxoids bystandard molecular techniques or by conjugation to a polysaccharide. Methods ofconjugation include those outlined in M. Brunswick et al., J. Immunol, 14023364(1988) and Chemistry of Protein Conjugates and Crosslinking, CRC Press, Boston(1991). Coupling of Shiga toxids to other proteins or polysaccharides would preventdisease from additional pathogens.Example VHis-Shiga ToxoidsA form of Shiga toxin that is immunoreactive but not toxinogenic is needed forimmunization in animals. Such a His-Shiga toxoid can be generated using chemicalor genetic methods. The chemical method involves treating the His-Shiga toxin witheither formaldehyde or glutaraldehyde, as described by Perera et al., J Clin. Microbiol.2612127 (1988)). Brieï¬y, samples of toxin containing 100 pg of protein are treated for3 days at 37°C with 0.1 M Na2HPO,, (pH 8.0) containing 1% formaldehyde, and theresidual formaldehyde is removed by dialysis against phosphate-buffered saline (PBS).To prepare His-Shiga toxoid by treatment with glutaraldehyde, crude toxin samplescontaining 50 pg of protein are incubated at 37°C in 0.11% glutaraldehyde in 0.1 MNa3HP04 (pH 8.0) for 30 min. The toxoid is then tested on Vero cells, as described inExample III, for loss of cytotoxicity.Genetically, a toxoid may be produced by siteâdirected mutagenesis, asdescribed in Gordon et al., Infect. Immun. 602485 (1992); Hovde et al., Proc. Natl.Acad. Sci. 85:2568 (1988); Jackson et al., J. Bacteriol. 172: 3346-3350 (1990).Several methods and kits exist for siteâdirected mutagenesis of a gene. One methodemploys the Bio-Rad Muta-Gene in vitro mutagenesis kit. Oligonucleotides can bedesigned and synthesized which alter speciï¬c condons in the toxin genes. Uracil-incorporated, single-stranded target plasmid DNA will be mutagenized according to10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/ 15836-19-the directions supplied by the manufacturer of the mutagenesis kit. The nucleotidechanges are then conï¬rmed by DNA sequence analysis.In the His-Shiga toxin, two or more amino acids essential for enzymatic activityshould be altered. For example, A subunit targets are the residues E167 and E170. TheShiga toxoid resulting from this mutation has been used for vaccinating pigs. (Gordonet al., supra).Example VIPassive Immunity to Shiga Toxin Using His-Shiga Toxin and ToxoidA. Antisera Specific for His-Shiga ToxinsAntisera speciï¬c for Shiga toxins are required to treat and prevent potentially-deadly infections by EHEC and Shigella dysenteriae type I. Speciï¬cally, once a childbecomes infected, the child and his or her family members or children in his or her daycare group can receive anti His-Shiga toxin sera to achieve a protective immuneresponse. A protective immune response is one that elicits sufï¬cient antibody topermit a patient to avoid infection, decrease the signiï¬cance or severity of an infection,or decrease the ability of bacteria to colonize the gastrointestinal tract.Animal studies have shown that administering anti-Shiga toxin sera to miceresults in resistance to normally lethal infection of EHEC. (Lindgren et al., Infect.Immun. 62:623(1994); Wadolowski et al., Infect. Immun. 5813959). Thus, applicantsbelieve that administering anti-Shiga toxin sera to humans and other mammals wouldresult in a protective immune response against Shiga toxin infections.Methods are wellâknown in the art for producing antisera for passiveimmunization. For example, His-Shiga toxoid, obtained by the methods described inExample VI, can be administered to a mammal, such as a horse intraperitoneally.Currently, the horse is used to produce serum against botulism toxin for administrationto humans, Hibbs et al., Clin. Infect Dis., 232337-40 (1996), and the horse would bea preferred method for producing shiga toxin antiserum. After several boosts with His-Shiga toxoid, the serum of the immunized horse (or other mammal) would be testedfor neutralizing the cytotoxicity of Shiga toxins. Advantageously, a large amount of10152025W0 98/11229CA 02265887 1999-03-09PCT/US97/15836-20-serum can be quickly made using this method. However, patients must first bescreened for an immune reaction to horse serum. For this purpose, a small amount ofhorse serum would be subcutaneously injected, and the patient would be monitored fora reaction. Such methods for administering horse antiserum against toxins to humansare well known to the skilled artisan. Hibbs et al., supra; Dehesa and Possani,Toxicon, 32: 1015-101 8(l 994); Gilan et al., Toxicon, 27:1 105-11 12 (1989).More preferably, the HisâShiga toxoid can be administered to humanvolunteers, either intraperitoneally or orally. The plasma from these volunteers is thenisolated, and the human anti-His-Shiga toxin serum can be administered to patients.No threat of serum sickness arises from this method. Human hyperimmune globulinto Hemophilus inï¬uenzae b, Streptococcus pneumoniae, and Neisseria meningitidishas previously been prepared by others (Siber et al., Infect. and Immun., 45: 248-254(1984)).B. Vaccines Against Shiga ToxinsAn embodiment of the invention is vaccines against Shiga toxin infection. Forexample, these vaccines can include antibodies directed against His-Shiga toxin,obtained further described in Example VII. Moreover, these vaccines can becombination vaccines that comprise His-Shiga toxoid fused or conjugated with anotherprotein, hapten, or antigen, as described in Example IV. These vaccines can beadministered intraperitoneally or injectably by methods well known in the art.A preferred method of administering HisâShiga toxin or toxoid and fusionsthereof is by further conjugation to Synsorb® (SynSorb Biotech, Inc., 1204Kensington Road, N.W., Calgary, Alberta, Canada, T2N3P5.) Synosorb is a sand-likematerial to which Shiga toxin receptor (Gb3) is covalently bound (Armstrong et al.,J. Infect. Dis., 171 : 1042 (1995)). This compound has been shown to bind Shiga toxinsand appears to be safe for human ingestion. (Armstrong et al., supra) The Synsorb isbound to the B subunit pentamer via the B subunit pentamer-receptor reaction.Conjugation with Synsorb adds further stability.10152025WO 98111229CA 02265887 1999-03-09PCT/US97l 15836-21-Another embodiment of the invention involves the administration of nucleicacid vaccines. DNA encoding a His-Shiga toxoid is injected into a patient as nakedDNA, or the DNA is delivered to the body by a carrier system such as retro viruses,adenoviruses, or other carriers known in the art. Following administration, the patientmounts an immune response against transiently expressed foreign antigens.Currently nucleic acid vaccines, in general, are all nearing clinical trials. Thisapproach to vaccines involves delivering the DNA encoding the desired antigen intothe host by inserting the gene into a nonreplicating plasmid vector (Marwick, C.JAMA 273: l403(1995); reviewed in Vogel, F.R. and N. Sarver, Clin. Microbiol, Rev.8:406 (1995)).The first published demonstration of the protective efï¬cacy of such a vaccinehas shown that intramuscular injection of plasmid DNA encoding inï¬uenza A virus(A/PR/8/34) nucleoprotein (NP) elicited protective immune responses in BALB/c miceagainst a heterologous strain of inï¬uenza virus (A/HK/68) (Ulmer, J .B. et al. Science259:] 745(l 993)). Immunized animals had reduced virus titers in their lungs,decreased weight loss, and increased survival compared with challenged control mice.Both NP-speciï¬c cytotoxic T lymphocytes (CTL's) and NP antibodies were generated.The NP antibodies were ineffective at conferring protection, but the CTL's killed virus-infected cells and cells pulsed with the appropriate major histocompatibility complexclass I-restricted peptide epitope.Another study has shown that intramuscular injection of plasmid DNAencoding inï¬uenza virus A/PR/8/ 34 hemagglutinin resulted in the generation ofneutralizing antibodies that protected mice against a heterologous lethal inï¬uenza viruschallenge (Montgomery, D.L. et al. DNA Cell Biol., l2:777 (1993)).Example VIIHis-Shiga AntibodiesHis-Shiga antibodies, polyclonal and monoclonal, can also be used in thetreatment, diagnosis, and prevention of infections related to Shiga toxins. Because oftheir increased speciï¬city, monoclonal antibodies are preferred. His-Shiga toxin10152025WO 98111229CA 02265887 1999-03-09PCT/US97/15836-22-antibodies can be administered to humans or other mammals to achieve a protectiveimmune response, for treatment or prophylaxis. Antibodies, in a physiologicallyacceptable carrier, may be administered orally or intraperitoneally. For this purpose,monoclonal antibodies are preferred and humanized monoclonal antibodies areparticularly preferred. Positive clinical responses in humans have been obtained withmonoclonal antibodies, and one skilled in the art would know how to employ Shigamonoclonal antibodies in humans. See Fagerberg et al., "Tumor Regression inMonoclonal Antibody-treated Patients Correlates with the Presence of Anti-idiotypeâreactive T Lymphocytes," Cancer Research, 55:1824â27 (1995); "A Phase I Study ofHuman/Mouse Chimeric Anti-ganglioside GD2 Antibody ch14.18 in Patients withNeuroblastoma," Eur. J. Cancer, 2:261-267 (1995)).Another embodiment of the invention involves using antibodies to diagnoseShiga toxin infections. The antibody, using well-known methods of immunoassaying,is brought into contact with a sample from a patient, such as a fecal sample. Inaddition, the antibody may be used to detect Shiga toxins in sample taken from cow,such as cow feces. Moreover, meat may be tested using the anti His-Shiga toxinantibody for detection. A detection kit comprising the His-Shiga toxin antibody canbe used for this purpose.For example, a sandwich Elisa can be used. In this kit, rabbit anti-His-Shigatoxin antibody can be used to capture toxin from a sample to be tested. Goat anti-His-Shiga toxin antibody can then be added followed by a secondary antibody such asmouse oz-goat antibody conjugated to horseradish peroxidase. The antibody can bedetected by standard methods.His-Shiga toxin polyclonal antibodies and monoclonal antibodies are describedbelow.A. Making Polyclonal AntibodiesThe technique of Harlow, E. and D. Lane (eds.), Antibodies- a LaboratoryManual, Cold Spring Harbor, New York (1988), may be followed. The generalprocedure is outlined herein. Take pre-bleeds of each mouse to be immunized: Bleed10152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97/ 15836-23-from the tail vein into an eppendorf tube. Incubate at 37°C for 30 min, stir gently witha sterile toothpick (to loosen the clot), store overnight at 4°C. In the morning, spin 10min/ 10,000 rpm in the microfuge, and collect the serum (i.e., supernatant; red bloodcells are the pellet). Store the serum at â20°C. The sera obtained will be used as anegative control after the mice are immunized.Inject a BALB/c mouse intraperitoneally with 25 pg of His-Shiga toxoid (usingTitremax adjuvant, according to the instructions of the manufacturer (CytRyx Corp.,154 Technology Pkwy., Norcross, GA. 30092, 800-345-2987)). Wait 2 weeks, boostwith an identical shot, wait 7 days and bleed from the tail vein into an eppendorf tube.Incubate at 37°C for 30 min, stir gently with a sterile toothpick (to loosen the clot),store overnight at 4°C. In the morning, spin 10 min/10.000 rpm in the microfuge, andcollect the serum. Store the sera at â20°C.B. ELISA to test titer of Abs.The technique of Harlow, E. and D. Lane (eds.), Antibodies: A LaboratoryManual, Cold Spring Harbor, New York (1988), may be followed. The generalprocedure is outlined below:(1) bind His-Shiga toxoid to plastic microtiter plates at 50 ng/well in PBS. Incubate2h/RT (room temp) or overnight at 4°C.(2) wash plate 2X with PBS.(3) block wells with 100 pl blocking solution [3% bovine serum albumin (SigmaChemical, St. Louis, MO.), 0.02% sodium azide (Sigma) in PBS - store stock at 4°C]forl-2hatRT.(4) wash plate 2X with PBS.(5) primary Ab = 50 pl test sera diluted in blocking solution for example, start with1:50 and do eleven 1:2 dilutions, or start with 1:50 and do eleven 1:10 dilutions),incubate 2 h/RT.(6) wash 4X with PBS.(7) secondary Ab = goat horseradish-conjugated anti-mouse lg, affinity puriï¬ed(Boehringer Mannheim Corp., 9115 Hague Rd., P.O. Box 50414, Indianapolis, IN.10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/15836-24-46250,800-262-1640). Add secondary Ab diluted 1:500 in blocking solution withoutazide. Incubate 1 h/RT.(8) wash 4X with PBS.(9) add 100 pl TMB Peroxidase substrate to each well (prepared according to theinstructions of the manufacturer, BioRad Labs, 3300 Regatta Blvd., Richmond, CA.94804). Allow blue color to develop (no more than 10 min). Stop the reaction with100 pl HZSO4. Read the plate at 450 nm.A titer is deï¬ned as an absorbance value 20.2 units above that obtained formouse preimmune sera.Anti-Shiga toxin Abs obtained from animals may be used clinically if onechanges the speciï¬city of the antibody to htunan. Such techniques are well known tothose of ordinary skill in the art. G. Winter et al., "Man-made antibodies," Nature,349: 293-299 (1991); P.T. Jones et al., "Replacing the complementarity-deterrniningregions in a human antibody with those from a mouse," Nature, 321: 522-525 (1986);P. Carter et al., "Humanization of an anti-pl 85âER2 antibody for human cancertherapy," Proc. Natl. Acad Sci. USA, 89: 4285-4289 (1992). Such antibodies may begiven to the sibling of an infected patient to reduce the risk of infection of the sibling.C. Raising Monoclonal Antibodies to His-Shiga ToxinMonoclonal antibodies directed against Shiga toxin are used to passivelyprotect a patient against EHEC and Shigella dysenteriae type I infections. Monoclonalantibodies are generated from mouse cells, and the speciï¬city of these antibodies arechanged for use in humans. G. Winter et al., "Man-made antibodies," Nature, 349:293-299 (1991); P.T. Jones et al., "Replacing the complementarity-determining regionsin a human antibody with those from a mouse," Nature, 321:522-525 (1986); P. Carter et al., "Humanization of an antiâpl85"Eâ2 antibody for humancancer therapy," Proc. Natl. Acad Sci. USA, 89: 4285-4289 (1992). Monoclonal Absrepresent a more "pure" antibody for administration to a patient.The procedure outlined in Harlow, E. and D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor, New York (1988) is followed: Five 4- to 5-week oldl01520W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-25-female BALB/cJ mice are prebled, and immunized intraperitoneally with 25 pg His-Shiga toxoid suspended in 100 pl of TiterMax. Mice are boosted twice in two weekintervals, intraperitoneally with 25 pg His-Shiga toxoid suspended in 100 pl ofTiterMax. Seven days after each boost, blood (~3 00 - 500 pl) is collected from the tailvein. Sera are assayed for the presence of antiâShiga toxin antibody by ELISA (asdescribed above).Mice producing high titers of anti-His Shiga toxin antibodies are boosted bothintravenously and intraperitoneally with 25 pg of His-Shiga toxoid in 100 pl of PBS,sacriï¬ced three days later, and sera collected. Spleen cells are isolated and fused toSp2/O-Ag mouse myeloma cells (ATCC #CRLl58l) at a ratio of 10 spleen cells to lmyeloma cell. Fused cells are distributed into microdilution plates, and culturesupematants are assayed by ELISA after 3-4 weeks of culture for anti-His-Shiga toxinantibodies. Cultures positive for production of anti-His Shiga toxin antibodies areexpanded and cloned twice by limiting dilution.The person skilled in the art would understand how to use and practice theinvention based on the above disclosure. Other embodiments of the invention will beapparent to those skilled in the art from consideration of the speciï¬cation and practiceof the invention disclosed herein. It is intended that the speciï¬cation and examples beconsidered as exemplary only, with the true scope and spirit of the invention beingindicated by the following claims.
Claims (2)
1. A polypeptide comprising a Shiga toxin having a histidine tag.
2. A polypeptide comprising an immunoreactive but non-toxinogenic form of the polypeptide of claim 1.
3. A fusion protein comprising the polypeptide of claim 1 or 2 fused to a second polypeptide or a portion thereof.
4. A method for large-scale isolation and purification of Shiga toxin comprising the steps of:
a) expressing Shiga toxin with a histidine tag in bacteria; and b) eluting cell extract containing histidine-tagged Shiga toxin over a nickel-nitrilotriacetic acid ligand (Ni-NTA) gel.
5. A method of providing passive immune protection comprising the step of administering antisera directed against the polypeptide of claim 2 to patients in need thereof.
6. A method of treating infections mediated by toxins of the Shiga toxin family comprising the step of administering antibodies against the polypeptide of claim 2 to patients in need thereof.
7. A vaccine comprising an antibody directed against the polypeptide of claim 2.
8. A vaccine comprising a nucleotide encoding the polypeptide of
2. A polypeptide comprising an immunoreactive but non-toxinogenic form of the polypeptide of claim 1.
3. A fusion protein comprising the polypeptide of claim 1 or 2 fused to a second polypeptide or a portion thereof.
4. A method for large-scale isolation and purification of Shiga toxin comprising the steps of:
a) expressing Shiga toxin with a histidine tag in bacteria; and b) eluting cell extract containing histidine-tagged Shiga toxin over a nickel-nitrilotriacetic acid ligand (Ni-NTA) gel.
5. A method of providing passive immune protection comprising the step of administering antisera directed against the polypeptide of claim 2 to patients in need thereof.
6. A method of treating infections mediated by toxins of the Shiga toxin family comprising the step of administering antibodies against the polypeptide of claim 2 to patients in need thereof.
7. A vaccine comprising an antibody directed against the polypeptide of claim 2.
8. A vaccine comprising a nucleotide encoding the polypeptide of
claim 2.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US2563796P | 1996-09-10 | 1996-09-10 | |
US60/025,637 | 1996-09-10 | ||
PCT/US1997/015836 WO1998011229A2 (en) | 1996-09-10 | 1997-09-09 | Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2265887A1 true CA2265887A1 (en) | 1998-03-19 |
Family
ID=21827219
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002265887A Abandoned CA2265887A1 (en) | 1996-09-10 | 1997-09-09 | Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0929679A2 (en) |
JP (1) | JP2001500730A (en) |
AR (1) | AR010218A1 (en) |
AU (1) | AU4184597A (en) |
CA (1) | CA2265887A1 (en) |
WO (1) | WO1998011229A2 (en) |
Families Citing this family (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2766193B1 (en) | 1997-07-18 | 2001-09-14 | Inst Curie | CHEMICAL POLYPEPTIDE COMPRISING FRAGMENT B OF TOXIN SHIGA AND PEPTIDES OF THERAPEUTIC INTEREST |
GB9715177D0 (en) * | 1997-07-21 | 1997-09-24 | Neutec Pharma Plc | Medicament |
ATE310750T1 (en) * | 1998-05-15 | 2005-12-15 | Inst Curie | VEROTOXIN B SUBUNIT FOR IMMUNIZATION |
EP1057895A1 (en) * | 1999-06-04 | 2000-12-06 | Lohmann Animal Health GmbH & Co. KG | Fusion protein comprising Shiga toxin 2e B subunit, (vaccine)compositions comprising it, and methods for their production |
WO2007098201A2 (en) * | 2006-02-16 | 2007-08-30 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. | Shiga toxoid chimeric proteins |
JP2009536818A (en) * | 2006-04-20 | 2009-10-22 | ザ ヘンリー エム. ジャクソン ファウンデーション フォー ザ アドヴァンスメント オブ ミリタリー メディシン インコーポレイテッド | Methods and compositions based on Shiga toxic type 1 protein |
CN102292098A (en) | 2009-01-23 | 2011-12-21 | 杰克孙M.亨利基金会先进军事医学有限公司 | Methods and compositions based on Shiga toxin type 2 protein |
LT2970487T (en) * | 2013-03-12 | 2020-05-11 | Molecular Templates, Inc. | Cytotoxic proteins comprising cell-targeting binding regions and shiga toxin a subunit regions for selective killing of specific cell types |
ES2877356T3 (en) | 2014-01-27 | 2021-11-16 | Molecular Templates Inc | MHC class I epitope supplying polypeptides |
US11142584B2 (en) | 2014-03-11 | 2021-10-12 | Molecular Templates, Inc. | CD20-binding proteins comprising Shiga toxin A subunit effector regions for inducing cellular internalization and methods using same |
ES2723774T3 (en) * | 2014-03-11 | 2019-09-02 | Molecular Templates Inc | Proteins comprising binding regions, effector regions of the Shiga toxin subunit A and signal motifs of localization of carboxy terminal endoplasmic reticulum |
SI3604333T1 (en) * | 2014-03-11 | 2021-08-31 | Molecular Templates, Inc. | Proteins comprising amino-terminal proximal shiga toxin a subunit effector regions and cell-targeting immunoglobulin-type binding regions capable of specifically binding cd38 |
CN106604934A (en) | 2014-06-11 | 2017-04-26 | 分子模板公司 | Protease-cleavage resistant, shiga toxin subunit effector polypeptides and cell-targeted molecules comprising same |
AU2016215205B2 (en) | 2015-02-05 | 2021-10-21 | Molecular Templates, Inc. | Multivalent CD20-binding molecules comprising shiga toxin a subunit effector regions and enriched compositions thereof |
EP3660035A1 (en) | 2015-05-30 | 2020-06-03 | Molecular Templates, Inc. | De-immunized, shiga toxin a subunit scaffolds and cell-targeting molecules comprising the same |
KR102580647B1 (en) | 2016-12-07 | 2023-09-20 | 몰레큘러 템플레이츠, 인코퍼레이션. | Shiga toxin A subunit effector polypeptides, Shiga toxin effector scaffolds, and cell-targeting molecules for site-specific conjugation |
CN110536695B (en) | 2017-01-25 | 2024-05-10 | 分子模板公司 | Cell targeting molecules comprising deimmunized shiga toxin a subunit effectors and cd8+ T cell epitopes |
JP7323200B2 (en) | 2018-04-17 | 2023-08-08 | モレキュラー テンプレーツ,インク. | HER2 Targeting Molecules Containing Deimmunized Shiga Toxin A Subunit Scaffolds |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
HUT78048A (en) * | 1994-10-24 | 1999-07-28 | Ophidian Pharmaceuticals, Inc. | Vaccine and antitoxin for treatment and prevention of c. difficile disease |
CA2218601A1 (en) * | 1995-03-24 | 1996-10-03 | Ophidian Pharmaceuticals, Inc. | Treatment for verotoxin-producing escherichia coli |
-
1997
- 1997-09-09 JP JP10513748A patent/JP2001500730A/en active Pending
- 1997-09-09 AR ARP970104115A patent/AR010218A1/en unknown
- 1997-09-09 CA CA002265887A patent/CA2265887A1/en not_active Abandoned
- 1997-09-09 EP EP97939845A patent/EP0929679A2/en not_active Withdrawn
- 1997-09-09 WO PCT/US1997/015836 patent/WO1998011229A2/en not_active Application Discontinuation
- 1997-09-09 AU AU41845/97A patent/AU4184597A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP0929679A2 (en) | 1999-07-21 |
WO1998011229A2 (en) | 1998-03-19 |
JP2001500730A (en) | 2001-01-23 |
WO1998011229A3 (en) | 1998-04-16 |
AU4184597A (en) | 1998-04-02 |
AR010218A1 (en) | 2000-06-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2265887A1 (en) | Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof | |
JP3927233B2 (en) | Pneumococcal polysaccharide-recombinant pneumolysin conjugate vaccines for immunization against pneumococcal infection | |
JP3914484B2 (en) | Soluble recombinant botulinum toxin protein | |
Chen et al. | Evaluation of purified UspA from Moraxella catarrhalis as a vaccine in a murine model after active immunization | |
US20050244425A1 (en) | Histidine-tagged intimin and methods of using intimin to stimulate an immune response and as an antigen carrier with targeting capability | |
Wen et al. | Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge | |
US20030219457A1 (en) | Soluble recombinant botulinum toxins | |
US20020176863A1 (en) | Antigen of hybrid M protein and carrier for group A streptococcal vaccine | |
Konadu et al. | Syntheses and immunologic properties of Escherichia coli O157 O-specific polysaccharide and Shiga toxin 1 B subunit conjugates in mice | |
Guth et al. | Variation in chemical properties and antigenic determinants among type II heat-labile enterotoxins of Escherichia coli | |
NZ216162A (en) | Immunogenic complex,methods of preparation,and intermediate products including peptide and recombinant dna | |
Sparling | Bacterial virulence and pathogenesis: an overview | |
US5837825A (en) | Campylobacter jejuni flagellin/Escherichia coli LT-B fusion protein | |
Osek et al. | Protection against Vibrio cholerae El Tor infection by specific antibodies against mannose-binding hemagglutinin pili | |
Calderwood et al. | A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin IB subunit | |
Mahajan et al. | Isolation, characterization, and host-cell-binding properties of a cytotoxin from Campylobacter jejuni | |
US5955293A (en) | Assays for shiga toxin and shiga-like toxins | |
US6762295B2 (en) | Protective helicobacter antigens | |
JP4081140B2 (en) | Hybrid molecule of non-heat-stable enterotoxin and cholera toxin B subunit | |
Walia et al. | Purification and characterization of novel toxin produced by Vibrio cholerae O1 | |
CN108339115B (en) | Pneumococcal combined vaccine using recombinant carrier protein and preparation method thereof | |
US6015889A (en) | Protein rib, a cell surface protein that confers immunity to many strains of the group B streptococcus: process for purification of the protein, reagent kit and pharmaceutical composition | |
Lobeck et al. | Towards a recombinant vaccine against diphtheria toxin | |
EP0368819A1 (en) | Expression of the binding subunit of cholera toxin with the aid of foreign promoters and/or leader sequences | |
KR100829380B1 (en) | Fowl cholera vaccine composition comprising recombinant outer membrane protein h of pasteurella multocida |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |