CA2388955A1 - Recombinant fusion molecules - Google Patents
Recombinant fusion molecules Download PDFInfo
- Publication number
- CA2388955A1 CA2388955A1 CA002388955A CA2388955A CA2388955A1 CA 2388955 A1 CA2388955 A1 CA 2388955A1 CA 002388955 A CA002388955 A CA 002388955A CA 2388955 A CA2388955 A CA 2388955A CA 2388955 A1 CA2388955 A1 CA 2388955A1
- Authority
- CA
- Canada
- Prior art keywords
- constant region
- antibody
- antigen
- recombinant
- antibody molecule
- 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
- 230000004927 fusion Effects 0.000 title abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 77
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 66
- 238000000034 method Methods 0.000 claims abstract description 46
- 238000004519 manufacturing process Methods 0.000 claims abstract description 14
- 230000001965 increasing effect Effects 0.000 claims abstract description 6
- 239000000427 antigen Substances 0.000 claims description 96
- 108091007433 antigens Proteins 0.000 claims description 92
- 102000036639 antigens Human genes 0.000 claims description 92
- 241000196324 Embryophyta Species 0.000 claims description 66
- 235000018102 proteins Nutrition 0.000 claims description 64
- 102000037865 fusion proteins Human genes 0.000 claims description 33
- 108020001507 fusion proteins Proteins 0.000 claims description 33
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 25
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 22
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 17
- 230000009261 transgenic effect Effects 0.000 claims description 16
- 241000725303 Human immunodeficiency virus Species 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 13
- 108020004511 Recombinant DNA Proteins 0.000 claims description 12
- 108060003951 Immunoglobulin Proteins 0.000 claims description 11
- 102000018358 immunoglobulin Human genes 0.000 claims description 11
- 241000282414 Homo sapiens Species 0.000 claims description 10
- 238000003776 cleavage reaction Methods 0.000 claims description 9
- 230000007017 scission Effects 0.000 claims description 9
- 241000283690 Bos taurus Species 0.000 claims description 5
- 241000288906 Primates Species 0.000 claims description 5
- 241000282836 Camelus dromedarius Species 0.000 claims description 4
- 241000282465 Canis Species 0.000 claims description 4
- 241000282324 Felis Species 0.000 claims description 4
- 241000124008 Mammalia Species 0.000 claims description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 4
- 241000283984 Rodentia Species 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 4
- 229960005486 vaccine Drugs 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 101710091045 Envelope protein Proteins 0.000 claims description 2
- 101710188315 Protein X Proteins 0.000 claims description 2
- 235000021245 dietary protein Nutrition 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims 3
- 241000701447 unidentified baculovirus Species 0.000 claims 2
- 241000203069 Archaea Species 0.000 claims 1
- 241000238631 Hexapoda Species 0.000 claims 1
- 102100021696 Syncytin-1 Human genes 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 18
- 230000028993 immune response Effects 0.000 abstract description 6
- 108020004414 DNA Proteins 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 8
- 239000006137 Luria-Bertani broth Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000002649 immunization Methods 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 230000009466 transformation Effects 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 6
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 6
- 108091005804 Peptidases Proteins 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 230000002163 immunogen Effects 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 6
- 238000011069 regeneration method Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 241000711895 Bovine orthopneumovirus Species 0.000 description 5
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000003155 DNA primer Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 244000061176 Nicotiana tabacum Species 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000419 plant extract Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 229940118376 tetanus toxin Drugs 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 3
- 102100029727 Enteropeptidase Human genes 0.000 description 3
- 108010013369 Enteropeptidase Proteins 0.000 description 3
- 108010074860 Factor Xa Proteins 0.000 description 3
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 3
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 108090000190 Thrombin Proteins 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000024203 complement activation Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 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 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229960004072 thrombin Drugs 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- LWTDZKXXJRRKDG-KXBFYZLASA-N (-)-phaseollin Chemical compound C1OC2=CC(O)=CC=C2[C@H]2[C@@H]1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-KXBFYZLASA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 102100034349 Integrase Human genes 0.000 description 2
- 241000282560 Macaca mulatta Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 108010055044 Tetanus Toxin Proteins 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 239000001166 ammonium sulphate Substances 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 2
- 229960003669 carbenicillin Drugs 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000002361 compost Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 235000013861 fat-free Nutrition 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 210000001539 phagocyte Anatomy 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000013615 primer Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 239000012882 rooting medium Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 229960000268 spectinomycin Drugs 0.000 description 2
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- FRGKKTITADJNOE-UHFFFAOYSA-N sulfanyloxyethane Chemical compound CCOS FRGKKTITADJNOE-UHFFFAOYSA-N 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102100022987 Angiogenin Human genes 0.000 description 1
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000004510 Collagen Type VII Human genes 0.000 description 1
- 108010017377 Collagen Type VII Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010062580 Concanavalin A Proteins 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010066486 EGF Family of Proteins Proteins 0.000 description 1
- 102000018386 EGF Family of Proteins Human genes 0.000 description 1
- 102100039371 ER lumen protein-retaining receptor 1 Human genes 0.000 description 1
- 108010075944 Erythropoietin Receptors Proteins 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010071289 Factor XIII Proteins 0.000 description 1
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 241000447437 Gerreidae Species 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 102000018932 HSP70 Heat-Shock Proteins Human genes 0.000 description 1
- 108010027992 HSP70 Heat-Shock Proteins Proteins 0.000 description 1
- 101000812437 Homo sapiens ER lumen protein-retaining receptor 1 Proteins 0.000 description 1
- 108700029227 Immunoglobulin Light Chain Genes Proteins 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 1
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 206010065764 Mucosal infection Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 102000004459 Nitroreductase Human genes 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 101710163504 Phaseolin Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010069013 Phenylalanine Hydroxylase Proteins 0.000 description 1
- 102100038223 Phenylalanine-4-hydroxylase Human genes 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 108050002653 Retinoblastoma protein Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 241000723792 Tobacco etch virus Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 1
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 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
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 102000001307 androgen receptors Human genes 0.000 description 1
- 108010080146 androgen receptors Proteins 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 108010072788 angiogenin Proteins 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 229960004261 cefotaxime Drugs 0.000 description 1
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229940012444 factor xiii Drugs 0.000 description 1
- 235000021312 gluten Nutrition 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000011544 gradient gel Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- 239000008062 guanidine hydrochloride buffer Substances 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000016784 immunoglobulin production 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
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 108091022911 insulin-like growth factor binding Proteins 0.000 description 1
- 102000028416 insulin-like growth factor binding Human genes 0.000 description 1
- 229940028885 interleukin-4 Drugs 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229940079938 nitrocellulose Drugs 0.000 description 1
- 108020001162 nitroreductase Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940126578 oral vaccine Drugs 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- LWTDZKXXJRRKDG-UHFFFAOYSA-N phaseollin Natural products C1OC2=CC(O)=CC=C2C2C1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-UHFFFAOYSA-N 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 230000009696 proliferative response Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003019 stabilising effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
- C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
-
- 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
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/13—Immunoglobulins specific features characterized by their source of isolation or production isolated from plants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Diabetes (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Obesity (AREA)
- AIDS & HIV (AREA)
- Pulmonology (AREA)
- Oncology (AREA)
- Endocrinology (AREA)
- Emergency Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
Abstract
This invention relates to a method for increasing the production of a protein in a cell and more particular by the production in a plant cell. This invention also relates to recombinant immune fusion complexes and more particularly to their expression in plants. Specific constructs comprising gp 120 fused to the heavy chain constant region of an antibody or part thereof are disclosed as well as the use of such constructs in generating an immune response.
Description
Recombinant Fusion Molecules This invention relates to a method for increasing the production of a protein in a cell and more particular the production in a plant cell. This invention also relates to recombinant immune fusion complexes and more particularly to their expression in plants.
Recombinant proteins have been expressed in transgenic plants by many groups.
A
consistent problem is low levels of expression, which complicates purification and processing. Various approaches have been taken to overcome this, for example using different regulatory gene constructs or constructing synthetic genes, with limited success. Thus the levels of expression routinely reported are up to 0.5% total soluble protein, with many examples being less than 0.01%. One notable exception has been antibody expression in plants. Our experience here is that levels of 1% are normally expected, with 5-8% also achieved. The reason for these differences is unclear, but we believe it to be related to the inherent stability of antibody molecules in plants, as well as the manner in which these proteins are handled by the plant cell machinery.
Immune complexes comprise antibodies and antigens, and give rise to the classical immune response. Immune complexes are conventionally produced by mixing antigen with antibody and allowing the molecules to associate via the binding site of the antibody and the specific epitope of the antigen. The disadvantage of this process is that immune complexes in vitYO which require careful mixing of antigen with antibody at optimal concentrations.
The present invention provides a method of increasing the production of a protein in a cell comprising expressing recombinant DNA encoding a recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region.
For convenience, the term 'heavy chain constant region' will be used throughout the following description to refer either to the full length immunoglobulin heavy chain constant region or to a part (fragment) of this region, as the context permits. The constant region comprises a number of regions (domains) depending of the class of the antibody. For an IgG antibody the constant region comprises three regions (domains) known as CHl, CH2, and CH3; the CH3 domain being the C-terminal domain. Thus instead of using the full length constant region, truncated versions of this may be used comprising the conjoined CH1/CH2, CH2/CH3 or CHl/CH3 domains.
By using the high expression of the immunoglobulin, we have found it possible to drive up the expression levels of the recombinant fused protein.
The desired protein can be any protein including reporter molecules such as (3-galactosidase, luciferase and GFP. The desired protein may also be a selectable marker such as an antibiotic.
Preferably, the desired protein is a food protein such as a storage protein, i.e. phaseolin or wheat gluten.
It is also preferably that the desired protein is a therapeutic protein.
Therapeutically useful proteins include receptors, e.g. the cystic fibrosis receptor (CFTR), enzymes including prodrug activating enzymes, e.g. nitroreductases, ligands, regulatory factors, hormones, and structural proteins. Therapeutic proteins also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondria) proteins, secreted proteins, plasmalemma-associated proteins and serum proteins. The desired protein may also be selected from lipoproteins, glycoproteins and phosphoproteins, hormones, growth factors, enzymes, clotting factors such as Factor XIII and Factor IX
etc., apolipoproteins, receptors, erythropoietin,drugs, oncogenes and tumor suppressors.
Specific examples of these compounds include proinsulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding proteins, epidermal growth factors, angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor, vascular endothelial growth factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxylase, oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p~3 protein, Rb gene product, cytokine receptor, J
~J~ 0~/327~4 PCT/G~00/04226 I-1, IL-6; ITL-~, viral capsid protein, and other proteins of asefui significance ir~ the body. The desired protein, which can be fused, is only limited by the availability of the nucleic acid sequence encoding the protein or polypeptide to be incorporated.
One skilled in the art will readily recognise that as more proteins and polypeptides become identified they can be integrated into the recombinant fusion protein of the present invention.
It is particularly preferred that the desired protein is an antigen. The term "antigen" as used herein refers to any protein that gives rise to an immune response in an animal such as a mammal, preferably a human. Preferably, the term "antigen" as used herein refers to a protein that stimulates a series of reaction in an animal that are mediated by white blood cells including lymphocytes, neutrophils and monocytes. Preferred antigens include viral antigens, bacterial antigens, protozoal antigens, parasitic antigens, tumor antigens, and proteins from viral, bacterial and parasitic organisms which can be used to induce an immune response. It is also preferred that the antigen is not a toxin.
A toxin is defined herein as a protein which has a direct toxic effect on a cell and causes cell death. It is most preferred that the antigen is human immunodeficiency virus gp 120, the non-toxic C-tetanus toxin fragment or bovine respiratory syncytial virus (BRSV) F-protein.
The desired protein may be linked to the constant region or part thereof directly or indirectly through an intermediate peptide linker which may include a peptide cleavage site. This will allow separation of the desired protein from the constant region of part thereof (if required) for purification purposes. Attachment of the desired protein to the constant region or part thereof may be made via the C-terminus of the heavy chain or part thereof. Alternatively, the antigen may be fused to the antibody at a site within the constant region or part thereof.
The method of the present invention is applicable to the production of the desired protein in any eukaryotic cell. Preferably the desired protein is produced in a eukaryotic cell which is capable of producing immunoglobulins. More preferably the desired protein is produced in a mammalian cell and most preferably in a plant cell.
The method of the present invention has the benefit of allowing the production of a desired protein in a cell at a higher level than when the desired protein is not produced as part of a fusion protein comprising a constant region of a heavy chain of an antibody.
Desired proteins are generally expressed at low levels in transgenic plants, whereas antibody molecules usually accumulate to much higher levels. By genetically fusing the desired protein with a component of the antibody molecule, the antibody component acts as a carrier and stabilising molecule and allows much higher levels of production and accumulation of the desired protein.
The present invention also provides a first recombinant fusion protein comprising an antigen fused to the constant region of the heavy chain of an antibody or to a part of said constant region. The recombinant fusion protein is as defined above in connection with the method of the present invention, except that the fusion protein comprises an antigen as defined above.
The present invention also provides a second recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region, wherein the fusion protein does not comprise a functional antigen binding domain. The recombinant fusion protein is as defined above in connection with the method of the present invention, except that the fusion protein does not comprise a functional antigen binding domain.
A functional antigen binding domain is defined herein as the antibody domain which contacts and binds an antigen. The domain comprises the complementarity determining regions (CDRs) of the antibody, which are well known to those skilled in the art and can be easily identified. The domain is preferably the variable domain of the heavy and/or light chain of an antibody. The second recombinant fusion protein of the present invention can comprise an antibody which has had one or more of its variable domains removed. Alternatively, the second recombinant fusion protein of the present invention can comprise an antibody which has one or more non-functional variable domains. The variable domains can be made non-functional by deleting the CDRs. Other methods for producing an antibody with a non-functional antigen binding domain are well known to those skilled in the art.
The present invention also provides a recombinant antibody molecule having an 5 antigen fused to an antibody molecule, wherein the antibody molecule has affinity for the antigen. The recombinant antibody molecule of the present invention can form an immune complex by associating with other recombinant antibody molecules of the present invention through the antigen binding sites of the antibody. Such an immune complex resembles, but is not identical to, a classical immune complex. A
significant advantage of the immune complex formed from the recombinant antibody molecules of the present invention is that it is easy to prepare in contrast to conventional preparation of immune complexes in vitro which require careful mixing of antigen with antibody at optimal concentrations. In our case, the expression ratio of antibody to antigen molecules is generally fixed at 1:1, which optimises the potential for forming immune complexes.
The recombinant antibody molecule of the present invention is highly immunogenic, especially when it is forms an immune complex, and is suitable for both systemic and mucosal immunisation without the need for an adjuvant. Adjuvants are generally incorporated into vaccine compositions to improve the immune response. As the recombinant antibody molecules of the present invention are highly immunogenic, it is possible to obtain immunisation without the need of an adjuvant.
The immune complex formed using recombinant antibody molecules of the present invention will possess substantially the same enhanced immunogenic properties as an endogenously produced immune complex or a conventionally produced immune complex.
The recombinant antibody molecule of the present invention can comprise any antibody molecule provided it is has affinity for the antigen to which it is fused. The antibody molecule can be a complete monoclonal antibody or an antigen binding fragment thereof, such as a Fv, Fab or F(ab')~ fragment. Preferably the antibody molecule comprises a constant region of a heavy chain of an antibody.
To engineer genetic constructs encoding antigen-antibody fusion proteins, there is no restriction as to the species of either of these components. The invention is of especial interest in relation to human medicine but other primate and other animal antigen/antibody combinations may be used depending on the intended biological application of the product. For example the antigens and the antibody heavy and light chains may be of human, rodent, rabbit, bovine, ovine, caprine, fowl, canine, camel, feline or primate origin.
To illustrate the range of application of the invention three model antigens have been used, namely human immunodeficiency virus gp 120, the non-toxic C-tetanus toxin fragment and bovine respiratory syncytial virus (BRSV) F-protein, with their respective specific monoclonal antibodies. For each antigen, numerous constructs are possible, in which the antigen is fused to either the C-terminus of a monoclonal antibody (Mab) full length heavy chain, or to truncated heavy chain domains consisting of 1, 2 or 3 constant region domains. Alternatively, the antigen is fused to the constant region at a point within the constant region.
The present invention also provides a transgenic plant expressing a recombinant fusion protein comprising a desired protein fused to a constant region of a heavy chain of an antibody.
The present invention also provides a transgenic plant expressing the first or second recombinant protein of the present invention, or the recombinant antibody molecule of the present invention.
Methods for generating transgenic plants are well known to those skilled in the art.
The transgenic plants of the present invention may express a number of molecular forms of fusion protein for each antigen, permitting the selection of the correctly formed molecular form of fusion protein and its expression at the highest levels and to extract and purify the fusion protein. It is also possible to characterise the plant derived fusion proteins by analysis of assembled molecular forms, recognition by and affinity for a panel of antibodies.
The advances deriving from this work are the development of a new type of engineered molecule suitable for vaccination in a range of infectious diseases. By using three different antigens, we are able to determine the efficacy of this approach for a standard bacterial immunogen, the envelope protein of an important human virus and a transmembrane fusion protein of a cattle virus that causes severe mucosal infections.
The recombinant antibody molecule resulting from the fusion of an antigen with an antibody molecule has all the components required for immune complex mediated stimulation of an immune response and our approach offers a convenient method for ensuring antigen/antibody complexing. The recombinant antibody molecules of the 1 S present invention can also form larger immune complexes in planta (see Figure 1) which can be used for immunisation. Not all parts of the antibody molecule are essential, such as the sites for FcR binding and complement activation that reside in the constant domains of IgG antibodies. Thus we also include the use of truncated antibody molecules which lack either the Cy3 domain (that is not involved in antigen recognition), or the Cyl domain.
It is also possible to use the first recombinant fusion protein of the present invention or the second recombinant fusion protein of the present invention (wherein the desired protein is an antigen) for immunisation. In particular, a fusion protein comprising an antibody molecule which does not comprise a functional antigen binding domain and an antigen can be used to deliver the antigen to phagocytes and also to initiate complement activation. By excluding the requirement for antigen recognition, it is possible to help prevent the phenomenon of modulation of antigen processing that results from antibody masking of T cell epitopes, as the antigen will be a fusion protein with the constant region of an antibody.
The use of plants is ideally suited for the production of the recombinant fusion proteins of the present invention not only because of the potential requirement for large quantities for vaccination purposes, but also because those complexes that involve full length antibody, or assembly with light chain are not readily produced in most other S expression systems.
To engineer genetic constructs encoding antigen-antibody fusion proteins DNA
encoding an antigen or antigenic fragment may be amplified by PCR. The genes encoding the light and heavy chains of murine, human or other mammalian monoclonal antibodies specific for these antigens are also cloned. Using these DNA
sequences the gene constructs can be made encoding the desired recombinant fusion protein.
For example, the following gene constructs for each of the antigens can be prepared for plant transformation:
Genetic constructs, showing Ig domains that will be included in each immune complex construct.
A - Heavy chain I) Construct #1 Variable---Cyl---Cy2---Cy3---Linker peptide---antigen II) Construct #2 Variable---Cyl---Cy2---Linker peptide---antigen 111) Construct #3 Cy2---Cy3---Linker peptide---antigen N) Construct #4 Cy2---Linker peptide---antigen B - Light chain Variable---CL
These constructs are shown diagrammatically in Figure 1, which also shows the final assembly of products in plants.
The linker peptide as shown in Figure 2 may conveniently be (GGGGS)3. A
conventional 6xHistidine tag and a protease cleavage site (e.g. enterokinase, Factor Xa or thrombin) may also be introduced at the C-terminus of the antigen for purification purposes. In addition a further different protease cleavage site (e.g.
enterokinase, Factor Xa or thrombin) may also be introduced between the linker peptide and the antigen.
Additional or alternative peptide tags and protease cleavage sites may be incorporated.
The gene constructs are sequenced for confirmatory purposes and inserted into plant expression vectors for plant transformation.
To generate transgenic plants expressing the 12 (four for each antigen) constructs, Nicotiana tabacum is a convenient plant host using Agrobacterium mediated transformation. For constructs I and II, in order to generate the recombinant fusion proteins , the immunoglobulin light chain genes and the heavy chain constructs may be introduced into separate plant lines as shown in Figure 1. Following regeneration of these first generation transformants, the light chain and heavy chain transformed plants are cross-fertilised and the second generation plants screened for production of the final product. For constructs III and IV, there is no requirement for the light chain and the final product can be produced in the first generation transformed plants.
To generate stable homozygous transgenic plant lines, transgenic plants expressing correctly assembled products are used to generate homozygous plant stocks. The plants are grown to maturity, self fertilised, and the resulting seeds screened by back crossing with non-transformed plants to determine those that are homozygous. Further stocks can be generated by self fertilisation and stored as seeds.
To characterise each molecular form of plant product and to extract and purify material for further study, the primary plant transformants are screened to determine which types of products are expressed and assembled optimally. This investigation can be performed by Western blot analysis and ELISA of crude plant extracts, using a range of antisera and monoclonal antibodies that are commercially available. Extraction and purification of the selected recombinant products may be achieved by ammonium sulphate precipitation, followed by filtration and affinity chromatography using either the immunoglobulin regions or specific peptides as affinity tags. Further characterisation is performed to determine molecular structure, recognition of the antigen moiety by a panel of antibodies and binding affinity.
The Figures show:
Recombinant proteins have been expressed in transgenic plants by many groups.
A
consistent problem is low levels of expression, which complicates purification and processing. Various approaches have been taken to overcome this, for example using different regulatory gene constructs or constructing synthetic genes, with limited success. Thus the levels of expression routinely reported are up to 0.5% total soluble protein, with many examples being less than 0.01%. One notable exception has been antibody expression in plants. Our experience here is that levels of 1% are normally expected, with 5-8% also achieved. The reason for these differences is unclear, but we believe it to be related to the inherent stability of antibody molecules in plants, as well as the manner in which these proteins are handled by the plant cell machinery.
Immune complexes comprise antibodies and antigens, and give rise to the classical immune response. Immune complexes are conventionally produced by mixing antigen with antibody and allowing the molecules to associate via the binding site of the antibody and the specific epitope of the antigen. The disadvantage of this process is that immune complexes in vitYO which require careful mixing of antigen with antibody at optimal concentrations.
The present invention provides a method of increasing the production of a protein in a cell comprising expressing recombinant DNA encoding a recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region.
For convenience, the term 'heavy chain constant region' will be used throughout the following description to refer either to the full length immunoglobulin heavy chain constant region or to a part (fragment) of this region, as the context permits. The constant region comprises a number of regions (domains) depending of the class of the antibody. For an IgG antibody the constant region comprises three regions (domains) known as CHl, CH2, and CH3; the CH3 domain being the C-terminal domain. Thus instead of using the full length constant region, truncated versions of this may be used comprising the conjoined CH1/CH2, CH2/CH3 or CHl/CH3 domains.
By using the high expression of the immunoglobulin, we have found it possible to drive up the expression levels of the recombinant fused protein.
The desired protein can be any protein including reporter molecules such as (3-galactosidase, luciferase and GFP. The desired protein may also be a selectable marker such as an antibiotic.
Preferably, the desired protein is a food protein such as a storage protein, i.e. phaseolin or wheat gluten.
It is also preferably that the desired protein is a therapeutic protein.
Therapeutically useful proteins include receptors, e.g. the cystic fibrosis receptor (CFTR), enzymes including prodrug activating enzymes, e.g. nitroreductases, ligands, regulatory factors, hormones, and structural proteins. Therapeutic proteins also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondria) proteins, secreted proteins, plasmalemma-associated proteins and serum proteins. The desired protein may also be selected from lipoproteins, glycoproteins and phosphoproteins, hormones, growth factors, enzymes, clotting factors such as Factor XIII and Factor IX
etc., apolipoproteins, receptors, erythropoietin,drugs, oncogenes and tumor suppressors.
Specific examples of these compounds include proinsulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding proteins, epidermal growth factors, angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor, vascular endothelial growth factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxylase, oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p~3 protein, Rb gene product, cytokine receptor, J
~J~ 0~/327~4 PCT/G~00/04226 I-1, IL-6; ITL-~, viral capsid protein, and other proteins of asefui significance ir~ the body. The desired protein, which can be fused, is only limited by the availability of the nucleic acid sequence encoding the protein or polypeptide to be incorporated.
One skilled in the art will readily recognise that as more proteins and polypeptides become identified they can be integrated into the recombinant fusion protein of the present invention.
It is particularly preferred that the desired protein is an antigen. The term "antigen" as used herein refers to any protein that gives rise to an immune response in an animal such as a mammal, preferably a human. Preferably, the term "antigen" as used herein refers to a protein that stimulates a series of reaction in an animal that are mediated by white blood cells including lymphocytes, neutrophils and monocytes. Preferred antigens include viral antigens, bacterial antigens, protozoal antigens, parasitic antigens, tumor antigens, and proteins from viral, bacterial and parasitic organisms which can be used to induce an immune response. It is also preferred that the antigen is not a toxin.
A toxin is defined herein as a protein which has a direct toxic effect on a cell and causes cell death. It is most preferred that the antigen is human immunodeficiency virus gp 120, the non-toxic C-tetanus toxin fragment or bovine respiratory syncytial virus (BRSV) F-protein.
The desired protein may be linked to the constant region or part thereof directly or indirectly through an intermediate peptide linker which may include a peptide cleavage site. This will allow separation of the desired protein from the constant region of part thereof (if required) for purification purposes. Attachment of the desired protein to the constant region or part thereof may be made via the C-terminus of the heavy chain or part thereof. Alternatively, the antigen may be fused to the antibody at a site within the constant region or part thereof.
The method of the present invention is applicable to the production of the desired protein in any eukaryotic cell. Preferably the desired protein is produced in a eukaryotic cell which is capable of producing immunoglobulins. More preferably the desired protein is produced in a mammalian cell and most preferably in a plant cell.
The method of the present invention has the benefit of allowing the production of a desired protein in a cell at a higher level than when the desired protein is not produced as part of a fusion protein comprising a constant region of a heavy chain of an antibody.
Desired proteins are generally expressed at low levels in transgenic plants, whereas antibody molecules usually accumulate to much higher levels. By genetically fusing the desired protein with a component of the antibody molecule, the antibody component acts as a carrier and stabilising molecule and allows much higher levels of production and accumulation of the desired protein.
The present invention also provides a first recombinant fusion protein comprising an antigen fused to the constant region of the heavy chain of an antibody or to a part of said constant region. The recombinant fusion protein is as defined above in connection with the method of the present invention, except that the fusion protein comprises an antigen as defined above.
The present invention also provides a second recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region, wherein the fusion protein does not comprise a functional antigen binding domain. The recombinant fusion protein is as defined above in connection with the method of the present invention, except that the fusion protein does not comprise a functional antigen binding domain.
A functional antigen binding domain is defined herein as the antibody domain which contacts and binds an antigen. The domain comprises the complementarity determining regions (CDRs) of the antibody, which are well known to those skilled in the art and can be easily identified. The domain is preferably the variable domain of the heavy and/or light chain of an antibody. The second recombinant fusion protein of the present invention can comprise an antibody which has had one or more of its variable domains removed. Alternatively, the second recombinant fusion protein of the present invention can comprise an antibody which has one or more non-functional variable domains. The variable domains can be made non-functional by deleting the CDRs. Other methods for producing an antibody with a non-functional antigen binding domain are well known to those skilled in the art.
The present invention also provides a recombinant antibody molecule having an 5 antigen fused to an antibody molecule, wherein the antibody molecule has affinity for the antigen. The recombinant antibody molecule of the present invention can form an immune complex by associating with other recombinant antibody molecules of the present invention through the antigen binding sites of the antibody. Such an immune complex resembles, but is not identical to, a classical immune complex. A
significant advantage of the immune complex formed from the recombinant antibody molecules of the present invention is that it is easy to prepare in contrast to conventional preparation of immune complexes in vitro which require careful mixing of antigen with antibody at optimal concentrations. In our case, the expression ratio of antibody to antigen molecules is generally fixed at 1:1, which optimises the potential for forming immune complexes.
The recombinant antibody molecule of the present invention is highly immunogenic, especially when it is forms an immune complex, and is suitable for both systemic and mucosal immunisation without the need for an adjuvant. Adjuvants are generally incorporated into vaccine compositions to improve the immune response. As the recombinant antibody molecules of the present invention are highly immunogenic, it is possible to obtain immunisation without the need of an adjuvant.
The immune complex formed using recombinant antibody molecules of the present invention will possess substantially the same enhanced immunogenic properties as an endogenously produced immune complex or a conventionally produced immune complex.
The recombinant antibody molecule of the present invention can comprise any antibody molecule provided it is has affinity for the antigen to which it is fused. The antibody molecule can be a complete monoclonal antibody or an antigen binding fragment thereof, such as a Fv, Fab or F(ab')~ fragment. Preferably the antibody molecule comprises a constant region of a heavy chain of an antibody.
To engineer genetic constructs encoding antigen-antibody fusion proteins, there is no restriction as to the species of either of these components. The invention is of especial interest in relation to human medicine but other primate and other animal antigen/antibody combinations may be used depending on the intended biological application of the product. For example the antigens and the antibody heavy and light chains may be of human, rodent, rabbit, bovine, ovine, caprine, fowl, canine, camel, feline or primate origin.
To illustrate the range of application of the invention three model antigens have been used, namely human immunodeficiency virus gp 120, the non-toxic C-tetanus toxin fragment and bovine respiratory syncytial virus (BRSV) F-protein, with their respective specific monoclonal antibodies. For each antigen, numerous constructs are possible, in which the antigen is fused to either the C-terminus of a monoclonal antibody (Mab) full length heavy chain, or to truncated heavy chain domains consisting of 1, 2 or 3 constant region domains. Alternatively, the antigen is fused to the constant region at a point within the constant region.
The present invention also provides a transgenic plant expressing a recombinant fusion protein comprising a desired protein fused to a constant region of a heavy chain of an antibody.
The present invention also provides a transgenic plant expressing the first or second recombinant protein of the present invention, or the recombinant antibody molecule of the present invention.
Methods for generating transgenic plants are well known to those skilled in the art.
The transgenic plants of the present invention may express a number of molecular forms of fusion protein for each antigen, permitting the selection of the correctly formed molecular form of fusion protein and its expression at the highest levels and to extract and purify the fusion protein. It is also possible to characterise the plant derived fusion proteins by analysis of assembled molecular forms, recognition by and affinity for a panel of antibodies.
The advances deriving from this work are the development of a new type of engineered molecule suitable for vaccination in a range of infectious diseases. By using three different antigens, we are able to determine the efficacy of this approach for a standard bacterial immunogen, the envelope protein of an important human virus and a transmembrane fusion protein of a cattle virus that causes severe mucosal infections.
The recombinant antibody molecule resulting from the fusion of an antigen with an antibody molecule has all the components required for immune complex mediated stimulation of an immune response and our approach offers a convenient method for ensuring antigen/antibody complexing. The recombinant antibody molecules of the 1 S present invention can also form larger immune complexes in planta (see Figure 1) which can be used for immunisation. Not all parts of the antibody molecule are essential, such as the sites for FcR binding and complement activation that reside in the constant domains of IgG antibodies. Thus we also include the use of truncated antibody molecules which lack either the Cy3 domain (that is not involved in antigen recognition), or the Cyl domain.
It is also possible to use the first recombinant fusion protein of the present invention or the second recombinant fusion protein of the present invention (wherein the desired protein is an antigen) for immunisation. In particular, a fusion protein comprising an antibody molecule which does not comprise a functional antigen binding domain and an antigen can be used to deliver the antigen to phagocytes and also to initiate complement activation. By excluding the requirement for antigen recognition, it is possible to help prevent the phenomenon of modulation of antigen processing that results from antibody masking of T cell epitopes, as the antigen will be a fusion protein with the constant region of an antibody.
The use of plants is ideally suited for the production of the recombinant fusion proteins of the present invention not only because of the potential requirement for large quantities for vaccination purposes, but also because those complexes that involve full length antibody, or assembly with light chain are not readily produced in most other S expression systems.
To engineer genetic constructs encoding antigen-antibody fusion proteins DNA
encoding an antigen or antigenic fragment may be amplified by PCR. The genes encoding the light and heavy chains of murine, human or other mammalian monoclonal antibodies specific for these antigens are also cloned. Using these DNA
sequences the gene constructs can be made encoding the desired recombinant fusion protein.
For example, the following gene constructs for each of the antigens can be prepared for plant transformation:
Genetic constructs, showing Ig domains that will be included in each immune complex construct.
A - Heavy chain I) Construct #1 Variable---Cyl---Cy2---Cy3---Linker peptide---antigen II) Construct #2 Variable---Cyl---Cy2---Linker peptide---antigen 111) Construct #3 Cy2---Cy3---Linker peptide---antigen N) Construct #4 Cy2---Linker peptide---antigen B - Light chain Variable---CL
These constructs are shown diagrammatically in Figure 1, which also shows the final assembly of products in plants.
The linker peptide as shown in Figure 2 may conveniently be (GGGGS)3. A
conventional 6xHistidine tag and a protease cleavage site (e.g. enterokinase, Factor Xa or thrombin) may also be introduced at the C-terminus of the antigen for purification purposes. In addition a further different protease cleavage site (e.g.
enterokinase, Factor Xa or thrombin) may also be introduced between the linker peptide and the antigen.
Additional or alternative peptide tags and protease cleavage sites may be incorporated.
The gene constructs are sequenced for confirmatory purposes and inserted into plant expression vectors for plant transformation.
To generate transgenic plants expressing the 12 (four for each antigen) constructs, Nicotiana tabacum is a convenient plant host using Agrobacterium mediated transformation. For constructs I and II, in order to generate the recombinant fusion proteins , the immunoglobulin light chain genes and the heavy chain constructs may be introduced into separate plant lines as shown in Figure 1. Following regeneration of these first generation transformants, the light chain and heavy chain transformed plants are cross-fertilised and the second generation plants screened for production of the final product. For constructs III and IV, there is no requirement for the light chain and the final product can be produced in the first generation transformed plants.
To generate stable homozygous transgenic plant lines, transgenic plants expressing correctly assembled products are used to generate homozygous plant stocks. The plants are grown to maturity, self fertilised, and the resulting seeds screened by back crossing with non-transformed plants to determine those that are homozygous. Further stocks can be generated by self fertilisation and stored as seeds.
To characterise each molecular form of plant product and to extract and purify material for further study, the primary plant transformants are screened to determine which types of products are expressed and assembled optimally. This investigation can be performed by Western blot analysis and ELISA of crude plant extracts, using a range of antisera and monoclonal antibodies that are commercially available. Extraction and purification of the selected recombinant products may be achieved by ammonium sulphate precipitation, followed by filtration and affinity chromatography using either the immunoglobulin regions or specific peptides as affinity tags. Further characterisation is performed to determine molecular structure, recognition of the antigen moiety by a panel of antibodies and binding affinity.
The Figures show:
Figure 1 shows schematically immune complexes in plants and potential assembly arrangements.
Figure 2 shows schematically the basic molecular design.
Figure 3 shows schematically the pEXGl3 principle cloning sites.
Figure 4 shows RT-PCR analysis of transgenic plants.
Figure 5 shows results of a capture ELISA for CH2-CH3-gp120.
Figure 6 shows results of a capture ELISA for recombinant gp120 expressed in plants Figure 7 shows a 10% SDS gel under reducing and non-reducing conditions.
EXAMPLE
Production and use of recombinant fusion molecules comprising gp120 and constant domains of a heavy chain of an antibody.
Cloning and genetic enaineerin~of HIV gp120 constructs:
The source of DNA for the HIV gp120 antigen was an infectious cloned isolate of HIV
IIIB. Such DNA can be obtained from the Medical Research Council AIDS Reagents Project (NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK).
The genes encoding the heavy and light chains of an HIV gp120 specific monoclonal antibody (e.g. Gorny et al., 1991, Proceedings of the National Academy of Sciences, USA 88:3238-3242) were cloned as described (Ma, J. K-C., T. Lehner, P.
Stabila, C.I.
Fux and A. Hiatt. 1994 Assembly of monoclonal antibodies with IgGl and IgA
heavy chain domains in transgenic tobacco plants. Eur. J. Immunol., 24:131-138). RNA
was extracted from relevant hybridoma cells using a standard extraction kit (e.g.
Promega SV Total RNA isolation system), cDNA was prepared by using the reverse transcriptase reaction. DNA encoding the antigen, or the heavy and light chains of a monoclonal antibodies specific to the antigen, or fragments of these antibody sequences, was amplified by the polymerase chain reaction using synthetic oligonucleotides shown in Figure 2. The oligonucleotide primers 1 and 2 corresponded to the 5' and 3' ends of the antibody heavy chain and the oligonucleotide primers 3 and 4 corresponded to the 5' and 3' ends of the antigen. The 5' primer for the antigen also includes sequences encoding a linker peptide and a protease cleavage site. The primers included appropriate restriction enzyme sites (such as 5' BamHI and 3' XmaI for gp120;
5' XhoI
and 3' BamHI for Ig heavy chain) and extra sequences encoding linker peptides (GGGSGGGSGGGS) or protease cleavage sites (e.g. Factor Xa IEGR). If a light chain is required, the gene can be cloned according to the methodology described by Drake et al., Antibody production in plants. P. Shepherd and Dean (eds). Monoclonal Antibodies - A practical approach. Oxford University Press. 2000) Engineering of DNA constructs was carned out in standard cloning vectors -pBluescript (Stratagene) and pET32 (New England Biolabs) by standard techniques as described in Maniatis, Fritsch and Sambrook (1982) - Molecular Cloning, A
Laboratory Manual. Essentially, the DNA encoding the antigen was first cloned into the vector using the multiple cloning site, followed by the DNA encoding the heavy and optionally the light chain, of an antibody or antibody fragment. In some cases, the order was reversed, with antibody genes being cloned first. Positive transformants were identified by the release of a DNA fragment of the correct size following digestion with the appropriate restriction enzymes, or PCR using the appropriate 5' and 3' oligonucleotide primers. Four chimeric immunoglobulin heavy chains constructs were engineered in which the antigen was expressed in fusion with varying portions of the immunoglobulin heavy chain as shown in Figure 1.
The specific example of cloning the Ig heavy chain CH2-CH3 domain fusion with an HIV gp120 fragment (construct III in Figure 1) is shown in Figure 2. Two PCR
products were amplified using oligonucleotide pairs 1-2 and 3-4. The HIV gp120 DNA
sequence encodes a truncated peptide starting at the N-terminus, up to and including the sequence KEYAL (aa147) with a stop codon immediately after. These were cloned into a vector based on pET32 which had previously been engineered with the key features of the cloning site within this vector shown in Figure 3. The gp 120 gene fragment was ligated into the BamHI - XmaI site of the vector, then the Ig heavy chain gene was cloned upstream in the NcoI - BamHI site. The entire construct was then re-amplified by PCR to include the downstream enterokinase cleavage site and His tag, and to include 5' XhoI and 3' EcoRI terminal restriction sites. This fragment was cloned into pBluescript for confirmatory sequencing.
The completed genetic constructs were then excised and cloned separately into a plant expression cassette such as that described above (Drake et al., 2000), within a plasmid (pMON530) suitable for initial screening in E. coli. Plasmids of this type are widely available, such as the pGreen system (www.p~reen.ac.uk). The plant expression cassette in this case contains the 5' untranslated region from tobacco etch virus which stabilises mRNA in plant cells, as well as an upstream marine IgG leader sequence (Figure 3), which directs secretion of the recombinant protein out of the plant cells.
This recombinant vector was used to transform E. coli (DHS-a). Screening of transformed clones was by Southern blotting, using radiolabelled DNA probes derived from the original PCR products. Plasmid DNA was purified from positive transformants (PromegaQiaprep kit) and used for transformation of Agrobacterium tumefaciens (strain LBA4404 - GibcoBRL, UK) using a freeze/thaw procedure as follows:
1. Inoculate 50 ml Luria broth (LB) containing spectinomycin (SO~g/ml) (Sigma) with Agrobacterium.
2. Shake at 28°C in the dark to an OD600 of 1Ø
3. Centrifuge the agrobacteria in sterile tubes at 3000g for 15 min.
4. Remove supernatant and, while keeping the tubes on ice, resuspend the bacterial pellet in a total of 1 ml of ice-cold l OmM CaClz.
5. Transfer 100 ml aliquots of the bacterial resuspension into sterile microcentrifuge tubes and then place into liquid nitrogen.
S 6. Use the frozen agrobacteria directly for DNA transformation or store at-80°C.
7. Pipette 10 ml of binary plasmid from a DNA miniprep onto the surface of 100 ml competent frozen agrobacteria.
8. Incubate the DNA-bacteria mixture for 5 min at 37 ° C in a water bath.
9. Add lml of LB to the bacteria and shake at 28°C for 4 h.
10. Centrifuge the bacteria for 2 min at 12,000 x g and resuspend the pellet in 100 ml of LB.
11. Spread 50 ml of the bacterial suspension onto LB medium (made semi-solid with 15 g1-' agar) containing spectinomycin required for selection of agrobacteria carrying binary and disarmed Ti helper plasmid. Seal the plates and incubate for 2-3 days at 28°C.
12. Re-streak resulting colonies on selective LB medium in separate Petri dishes and incubate at 28°C for 2 days.
Plant transformation and regeneration: All gene constructs were introduced into Nicotiana tabacum, var. xanthii. Tobacco transformation with A. tumefaciens was by standard procedures. Leaf discs are cut from surface sterilised tobacco leaves and incubated with a culture of the recombinant A. tumefaciens, containing cDNA
inserts.
The infected discs are transferred to culture plates containing a medium that induces regeneration of shoots, supplemented with kanamycin and carbenicillin (Sigma, UK).
Shoots developing after this stage are excised and transplanted onto a root inducing medium, supplemented with kanamycin. Rooted plantlets are transplanted into soil after the appearance of roots. The detailed methodology was as follows:
1. Remove Agrobacterium (containing binary vector) from -80°C and streak onto semi-solid LB medium (with appropriate antibiotics) in a 9 cm Petri dish.
Incubate at 28°C for 2 d.
2. Inoculate Agrobacterium from Petri dish into 10 ml LB.
3. Shake at 28°C to an OD600 of 1Ø
4. Remove 4 ml of Agrobacterium suspension and add to 16 ml of sterile distilled water in a sterile 9 cm Petri dish.
5. Cut 0.5 - 1.0 cm leaf discs from surface sterilised leaves and immerse for 5 min in the diluted Agrobacterium suspension.
Figure 2 shows schematically the basic molecular design.
Figure 3 shows schematically the pEXGl3 principle cloning sites.
Figure 4 shows RT-PCR analysis of transgenic plants.
Figure 5 shows results of a capture ELISA for CH2-CH3-gp120.
Figure 6 shows results of a capture ELISA for recombinant gp120 expressed in plants Figure 7 shows a 10% SDS gel under reducing and non-reducing conditions.
EXAMPLE
Production and use of recombinant fusion molecules comprising gp120 and constant domains of a heavy chain of an antibody.
Cloning and genetic enaineerin~of HIV gp120 constructs:
The source of DNA for the HIV gp120 antigen was an infectious cloned isolate of HIV
IIIB. Such DNA can be obtained from the Medical Research Council AIDS Reagents Project (NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK).
The genes encoding the heavy and light chains of an HIV gp120 specific monoclonal antibody (e.g. Gorny et al., 1991, Proceedings of the National Academy of Sciences, USA 88:3238-3242) were cloned as described (Ma, J. K-C., T. Lehner, P.
Stabila, C.I.
Fux and A. Hiatt. 1994 Assembly of monoclonal antibodies with IgGl and IgA
heavy chain domains in transgenic tobacco plants. Eur. J. Immunol., 24:131-138). RNA
was extracted from relevant hybridoma cells using a standard extraction kit (e.g.
Promega SV Total RNA isolation system), cDNA was prepared by using the reverse transcriptase reaction. DNA encoding the antigen, or the heavy and light chains of a monoclonal antibodies specific to the antigen, or fragments of these antibody sequences, was amplified by the polymerase chain reaction using synthetic oligonucleotides shown in Figure 2. The oligonucleotide primers 1 and 2 corresponded to the 5' and 3' ends of the antibody heavy chain and the oligonucleotide primers 3 and 4 corresponded to the 5' and 3' ends of the antigen. The 5' primer for the antigen also includes sequences encoding a linker peptide and a protease cleavage site. The primers included appropriate restriction enzyme sites (such as 5' BamHI and 3' XmaI for gp120;
5' XhoI
and 3' BamHI for Ig heavy chain) and extra sequences encoding linker peptides (GGGSGGGSGGGS) or protease cleavage sites (e.g. Factor Xa IEGR). If a light chain is required, the gene can be cloned according to the methodology described by Drake et al., Antibody production in plants. P. Shepherd and Dean (eds). Monoclonal Antibodies - A practical approach. Oxford University Press. 2000) Engineering of DNA constructs was carned out in standard cloning vectors -pBluescript (Stratagene) and pET32 (New England Biolabs) by standard techniques as described in Maniatis, Fritsch and Sambrook (1982) - Molecular Cloning, A
Laboratory Manual. Essentially, the DNA encoding the antigen was first cloned into the vector using the multiple cloning site, followed by the DNA encoding the heavy and optionally the light chain, of an antibody or antibody fragment. In some cases, the order was reversed, with antibody genes being cloned first. Positive transformants were identified by the release of a DNA fragment of the correct size following digestion with the appropriate restriction enzymes, or PCR using the appropriate 5' and 3' oligonucleotide primers. Four chimeric immunoglobulin heavy chains constructs were engineered in which the antigen was expressed in fusion with varying portions of the immunoglobulin heavy chain as shown in Figure 1.
The specific example of cloning the Ig heavy chain CH2-CH3 domain fusion with an HIV gp120 fragment (construct III in Figure 1) is shown in Figure 2. Two PCR
products were amplified using oligonucleotide pairs 1-2 and 3-4. The HIV gp120 DNA
sequence encodes a truncated peptide starting at the N-terminus, up to and including the sequence KEYAL (aa147) with a stop codon immediately after. These were cloned into a vector based on pET32 which had previously been engineered with the key features of the cloning site within this vector shown in Figure 3. The gp 120 gene fragment was ligated into the BamHI - XmaI site of the vector, then the Ig heavy chain gene was cloned upstream in the NcoI - BamHI site. The entire construct was then re-amplified by PCR to include the downstream enterokinase cleavage site and His tag, and to include 5' XhoI and 3' EcoRI terminal restriction sites. This fragment was cloned into pBluescript for confirmatory sequencing.
The completed genetic constructs were then excised and cloned separately into a plant expression cassette such as that described above (Drake et al., 2000), within a plasmid (pMON530) suitable for initial screening in E. coli. Plasmids of this type are widely available, such as the pGreen system (www.p~reen.ac.uk). The plant expression cassette in this case contains the 5' untranslated region from tobacco etch virus which stabilises mRNA in plant cells, as well as an upstream marine IgG leader sequence (Figure 3), which directs secretion of the recombinant protein out of the plant cells.
This recombinant vector was used to transform E. coli (DHS-a). Screening of transformed clones was by Southern blotting, using radiolabelled DNA probes derived from the original PCR products. Plasmid DNA was purified from positive transformants (PromegaQiaprep kit) and used for transformation of Agrobacterium tumefaciens (strain LBA4404 - GibcoBRL, UK) using a freeze/thaw procedure as follows:
1. Inoculate 50 ml Luria broth (LB) containing spectinomycin (SO~g/ml) (Sigma) with Agrobacterium.
2. Shake at 28°C in the dark to an OD600 of 1Ø
3. Centrifuge the agrobacteria in sterile tubes at 3000g for 15 min.
4. Remove supernatant and, while keeping the tubes on ice, resuspend the bacterial pellet in a total of 1 ml of ice-cold l OmM CaClz.
5. Transfer 100 ml aliquots of the bacterial resuspension into sterile microcentrifuge tubes and then place into liquid nitrogen.
S 6. Use the frozen agrobacteria directly for DNA transformation or store at-80°C.
7. Pipette 10 ml of binary plasmid from a DNA miniprep onto the surface of 100 ml competent frozen agrobacteria.
8. Incubate the DNA-bacteria mixture for 5 min at 37 ° C in a water bath.
9. Add lml of LB to the bacteria and shake at 28°C for 4 h.
10. Centrifuge the bacteria for 2 min at 12,000 x g and resuspend the pellet in 100 ml of LB.
11. Spread 50 ml of the bacterial suspension onto LB medium (made semi-solid with 15 g1-' agar) containing spectinomycin required for selection of agrobacteria carrying binary and disarmed Ti helper plasmid. Seal the plates and incubate for 2-3 days at 28°C.
12. Re-streak resulting colonies on selective LB medium in separate Petri dishes and incubate at 28°C for 2 days.
Plant transformation and regeneration: All gene constructs were introduced into Nicotiana tabacum, var. xanthii. Tobacco transformation with A. tumefaciens was by standard procedures. Leaf discs are cut from surface sterilised tobacco leaves and incubated with a culture of the recombinant A. tumefaciens, containing cDNA
inserts.
The infected discs are transferred to culture plates containing a medium that induces regeneration of shoots, supplemented with kanamycin and carbenicillin (Sigma, UK).
Shoots developing after this stage are excised and transplanted onto a root inducing medium, supplemented with kanamycin. Rooted plantlets are transplanted into soil after the appearance of roots. The detailed methodology was as follows:
1. Remove Agrobacterium (containing binary vector) from -80°C and streak onto semi-solid LB medium (with appropriate antibiotics) in a 9 cm Petri dish.
Incubate at 28°C for 2 d.
2. Inoculate Agrobacterium from Petri dish into 10 ml LB.
3. Shake at 28°C to an OD600 of 1Ø
4. Remove 4 ml of Agrobacterium suspension and add to 16 ml of sterile distilled water in a sterile 9 cm Petri dish.
5. Cut 0.5 - 1.0 cm leaf discs from surface sterilised leaves and immerse for 5 min in the diluted Agrobacterium suspension.
6. Briefly dry leaf discs on sterile filter paper.
7. Place leaf discs on shoot regeneration medium, 10-15 discs per 20 ml of medium in each 9 cm Petri dish. Incubate for 2 d at 25°C with a 16 h photoperiod.
8. Transfer leaf discs to shoot regeneration medium containing 500 mg/1 carbenicillin and appropriate concentration of selective agent for transformed plant cells.
Incubate for 21 d at 25°C with a 16 h photoperiod.
Incubate for 21 d at 25°C with a 16 h photoperiod.
9. Transfer leaf discs to shoot regeneration medium in sterile 175 ml glass jars containing 500 mg/1 cefotaxime and 200 mg/1 kanamycin. Incubate at 25°C
with a 16 h photoperiod.
with a 16 h photoperiod.
10. Developing shoots are removed when they reach a convenient size (approx.
0.5 cm in length) and transferred to rooting medium (3-4 shoots/40 ml medium/175 ml glass jar). Incubate for 14 d at 25°C with a 16 h photoperiod.
0.5 cm in length) and transferred to rooting medium (3-4 shoots/40 ml medium/175 ml glass jar). Incubate for 14 d at 25°C with a 16 h photoperiod.
11. Shoots lacking roots are trimmed at the base and replaced in fresh rooting medium for an additional 21 d or until roots appear.
12. Rooted shoots are transferred to compost in plant pots, watered and supplied with nutrients. Plants are kept in seed trays and covered with a lid for 24 h after transfer to compost, to minimise initial water loss.
Regenerated plants were screened for expression of immunoglobulin chains and each of the antigens by Western blot and ELISA of crude leaf extracts using available antisera and monoclonal antibodies (e.g. from the MRC AmS Directed reagents programme, address given above). Transgenic plants were self fertilised to establish homozygous plant lines and cross fertilised to generate antibody producing plants.
RNA extraction:
Total RNA was extracted from plants less than a month old, using the Promega plant RNA extraction kit (Rneasy Plant mini kit). Alternatively, leaf tissue was frozen in liquid nitrogen and rapidly ground to a fine powder. This was mixed with 2 volumes of ice cold guanidine hydrochloride buffer (8M guanidine hydrochloride , 20mM
MES, 20mM EDTA, 50mM (3-mercaptoethanol, pH7). After agitation it was added to 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1), mixed thoroughly, and 5 centrifuged at 10,000 rpm for 45 minutes. The upper (aqueous) phase was collected, mixed with pre-cooled ethanol (0.7 volumes) and 1M acetic acid (0.2 volumes), and incubated at -200°C for 16 hours. After centrifugation, the precipitate was washed three times with 3M sodium acetate, pH5.2, and once with 70% ethanol. The pellet was dissolved in sterile RNAse free water (Sigma, UK) containing RNAse free DNAse 10 (Promega, UK), and incubated at 37°C for 1 hour, then at 70°C
for 5 minutes.
RNA concentration and purity were assessed using the GeneQuant II RNA/DNA
Calculator (Pharmacia BioTech, UK). RNA was stored in sterile RNAse free water at -20°C.
RT-PCR was performed using appropriate oligonucleotide primers to determine that the correct RNA transcript was being made by putative transformed plants.
Characterisation and selection of recombinant immune complexes from plants:
To confirm the expression of each engineered constructs in plants, leaf extracts were examined by ELISA and Western blot analysis. Samples were extracted in 150mM
NaCI and 20mM tris, pH8 (TBS) with leupeptin (lOmg/ml) (Calbiochem). Capture ELISA analyses were performed by incubating the plant extracts on microtiter plates that had been pre-coated with a monoclonal antibody specific for HIV gp120 (ADP401 from the MRC Aids Directed Program) and blocked with 5% non-fat dry milk in TBS.
After overnight incubation at 4°C, the plates were washed in TBS with 0.05% Tween 20, then incubated with either a horseradish peroxidase labelled Sheep anti-Mouse IgG
antiserum (The Binding Site, UK), or one of a panel of gp120 specific monoclonal antibodies (MRC Aids Directed Program), followed by the relevant horseradish peroxidase antiserum (The Binding Site, UK). The assay was developed using the TMB
substrate (Sigma, UK) and absorbance was read at 450nm.
For Western blot analyses, the plant extracts were boiled in 75mM tris-HCl (pH6.8) and 2% SDS under either non-reducing or reducing conditions (by addition of 0.1%
(3 mercaptoethanol). SDS-polyacrylamide gel electrophoresis (PAGE) in 10%
acrylamide or 4-20% gradient gels was performed. The gels were blotted onto nitro-cellulose paper and blocked in TBS containing 0.05% Tween 20, and 1% non-fat dry milk. The blots were then incubated in appropriate antiserum for 2 hours at 37°C. After washing, an appropriate second-layer alkaline phosphatase-conjugated antiserum was applied for 2 hours at 37°C. Antibody binding was detected by incubation with nitroblue tetrazolium (300mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (1 SOmg/ml) (Promega, UK).
Glycosylation is determined by Western blot, examining binding to lectins such as concanavalin A, or by using glycans specific antisera (kindly provided by Dr.
Loic Faye, University of Rouen). Functional studies of antigen binding affinity to available monoclonal antibodies may be performed using surface plasmon resonance techniques.
Recombinant.protein purification:
Purification was performed using a procedure which we have previously determined for IgG extraction from transgenic plants. Following initial precipitation from crude plant extract with ammonium sulphate, the recombinant antibody was concentrated by stirred cell filtration using a YM30 molecular weight cut-off filter (Amicon, UK).
Purification was by affinity chromatography using agarose coupled to anti-mouse or human IgG
antibodies (Sigma, UK) as the ligand. Elution was in O.1M glycine-HCl pH 2.5.
The recombinant heavy chains may also be designed to allow a further affinity purification step using the 6xHis fusion peptide tag with immobilised metal chelate affinity chromatography, which can subsequently be cleaved by thrombin.
The expression of these fusion proteins is not limited to plants, and could be earned out in any other eukaryotic cells, especially cells that can produce immunoglobulins such as mammalian cells.
The immunoglobulin chain sequences can be exchanged for sequences from other classes of immunoglobulins.
Other sequences can be incorporated into the construct in addition to the epitope tags and protease cleavage sites. For example, these could code for other functional regions, such as markers, enzyme activity, or sites for chemical coupling to other active molecules.
In addition to HIV gp120, two further examples have been developed. Similar constructs have been prepared using tetanus toxin non-toxic C-fragment (kindly donated by Dr. Neil Fairweather at Imperial College, London) and a tetanus toxin specific murine monoclonal antibody (kindly donated by Dr. Claus Koch at the State Serum Institute, Copenhagen). In addition, we have prepared corresponding constructs for the F-protein of bovine respiratory syncytial virus and a specific murine monoclonal antibody the genes for which were kindly provided by Dr. K.G. Madsen at the Danish Veterinary Institute for Virus Research.
Among the benefits of the invention, the following are noteworthy:-(i)Co-expression of antibody with antigen increases the levels of expression of antigen.
(ii)Antigen and whole antibody are genetically linked to form a type of immune complex. In other constructs, antigen and antibody fragments (that may or may not include the antigen binding site) are linked to form fusion proteins.
(iii)The recombinant antibody molecule and the antigen are automatically expressed at 1:1 ratio. In the case of constructs involving whole IgG, larger complexes may form spontaneously.
(iv)Antigen-antibody fusion molecules are easy to produce.
(v)The use of plants to make these molecules significantly reduces the cost, particularly if production is increased to agricultural scale.
(vi)For oral vaccines, consumption of edible transgenic plants offers an alternative, safe mode of delivery for immune complex vaccines.
(vii)There is potentially no need for addition of an adjuvant as the engineered molecule is already highly immunogenic. The CH2 and CH3 immunoglobulin domains include the site for complement activation and opsonisation of phagocytes Results:
Results have been obtained for two of the described constructs - CH2-gp120 and CH2-CH3-gp 120. RT-PCR confirmed that specific mRNA of the desired size was being made by transgenic plants (Figure 4).
Analysis by capture ELISA demonstrated that both constructs are being expressed by plants. We have gone on to characterise CH2-CH3-gp120 more fully and demonstrated that by ELISA that it contains IgG2a epitopes as well as gp120 epitopes as shown in Figures 5 and 6. Moreover, expression of the recombinant gp 120 is greater in plants expressing the CH2-CH3-gp120 construct, than those expressing either gp120 alone, gp 120 with a C-terminal HDEL tag, or CH2-gp 120. Preliminary results indicate an expression level of gp120 of approximately 0.8%.
Purification of plant derived material has been carried out and Western blot analysis demonstrates the presence of protein bands that are recognised by specific antibodies to gp120 and murine IgG2a (Figure 7). Under reducing conditions, the most prominent band has a molecular size consistent with that expected of approximately SOKd.
Under non-reducing conditions, higher molecular weight forms approaching 100Kd are detected. This is consistent with the formation of homodimers, through association of the CH2 and CH3 domains of the heavy chain.
A preliminary investigation of immunogenicity has been carned out in a single rhesus monkey. Following 2 administrations of purified plant derived CH2-CH3-gp 120 (150~g per dose) covalently linked with the adjuvant mycobacterial heat shock protein 70 using the SPDP bifunctional reagent, specific antibody and cellular immune responses were detected. After 3 immunisations, very significant T-cell proliferative responses were detected, not only to the plant CH2-CH3-gp120 construct, but also to CA 02388955 2002-04-24' purified recombinant gp120 expressed in Chinese hamster ovary cells. This indicates that the plant recombinant gp120 is correctly expressed and folded in an immunogenic conformation. Furthermore, four macaques have been immunised by the mucosal intra-vaginal route, and four by the targeted iliac lymph node route (Lehner et al., Nature Medicine, 2:767-775, 1996). After two immunisations, induction of interferon-y and interleukin-4 has been detected, indicating that both THl and TH2 responses are elicited. Further immunisation studies are being carned out in rhesus monkeys as well as mice.
Regenerated plants were screened for expression of immunoglobulin chains and each of the antigens by Western blot and ELISA of crude leaf extracts using available antisera and monoclonal antibodies (e.g. from the MRC AmS Directed reagents programme, address given above). Transgenic plants were self fertilised to establish homozygous plant lines and cross fertilised to generate antibody producing plants.
RNA extraction:
Total RNA was extracted from plants less than a month old, using the Promega plant RNA extraction kit (Rneasy Plant mini kit). Alternatively, leaf tissue was frozen in liquid nitrogen and rapidly ground to a fine powder. This was mixed with 2 volumes of ice cold guanidine hydrochloride buffer (8M guanidine hydrochloride , 20mM
MES, 20mM EDTA, 50mM (3-mercaptoethanol, pH7). After agitation it was added to 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1), mixed thoroughly, and 5 centrifuged at 10,000 rpm for 45 minutes. The upper (aqueous) phase was collected, mixed with pre-cooled ethanol (0.7 volumes) and 1M acetic acid (0.2 volumes), and incubated at -200°C for 16 hours. After centrifugation, the precipitate was washed three times with 3M sodium acetate, pH5.2, and once with 70% ethanol. The pellet was dissolved in sterile RNAse free water (Sigma, UK) containing RNAse free DNAse 10 (Promega, UK), and incubated at 37°C for 1 hour, then at 70°C
for 5 minutes.
RNA concentration and purity were assessed using the GeneQuant II RNA/DNA
Calculator (Pharmacia BioTech, UK). RNA was stored in sterile RNAse free water at -20°C.
RT-PCR was performed using appropriate oligonucleotide primers to determine that the correct RNA transcript was being made by putative transformed plants.
Characterisation and selection of recombinant immune complexes from plants:
To confirm the expression of each engineered constructs in plants, leaf extracts were examined by ELISA and Western blot analysis. Samples were extracted in 150mM
NaCI and 20mM tris, pH8 (TBS) with leupeptin (lOmg/ml) (Calbiochem). Capture ELISA analyses were performed by incubating the plant extracts on microtiter plates that had been pre-coated with a monoclonal antibody specific for HIV gp120 (ADP401 from the MRC Aids Directed Program) and blocked with 5% non-fat dry milk in TBS.
After overnight incubation at 4°C, the plates were washed in TBS with 0.05% Tween 20, then incubated with either a horseradish peroxidase labelled Sheep anti-Mouse IgG
antiserum (The Binding Site, UK), or one of a panel of gp120 specific monoclonal antibodies (MRC Aids Directed Program), followed by the relevant horseradish peroxidase antiserum (The Binding Site, UK). The assay was developed using the TMB
substrate (Sigma, UK) and absorbance was read at 450nm.
For Western blot analyses, the plant extracts were boiled in 75mM tris-HCl (pH6.8) and 2% SDS under either non-reducing or reducing conditions (by addition of 0.1%
(3 mercaptoethanol). SDS-polyacrylamide gel electrophoresis (PAGE) in 10%
acrylamide or 4-20% gradient gels was performed. The gels were blotted onto nitro-cellulose paper and blocked in TBS containing 0.05% Tween 20, and 1% non-fat dry milk. The blots were then incubated in appropriate antiserum for 2 hours at 37°C. After washing, an appropriate second-layer alkaline phosphatase-conjugated antiserum was applied for 2 hours at 37°C. Antibody binding was detected by incubation with nitroblue tetrazolium (300mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (1 SOmg/ml) (Promega, UK).
Glycosylation is determined by Western blot, examining binding to lectins such as concanavalin A, or by using glycans specific antisera (kindly provided by Dr.
Loic Faye, University of Rouen). Functional studies of antigen binding affinity to available monoclonal antibodies may be performed using surface plasmon resonance techniques.
Recombinant.protein purification:
Purification was performed using a procedure which we have previously determined for IgG extraction from transgenic plants. Following initial precipitation from crude plant extract with ammonium sulphate, the recombinant antibody was concentrated by stirred cell filtration using a YM30 molecular weight cut-off filter (Amicon, UK).
Purification was by affinity chromatography using agarose coupled to anti-mouse or human IgG
antibodies (Sigma, UK) as the ligand. Elution was in O.1M glycine-HCl pH 2.5.
The recombinant heavy chains may also be designed to allow a further affinity purification step using the 6xHis fusion peptide tag with immobilised metal chelate affinity chromatography, which can subsequently be cleaved by thrombin.
The expression of these fusion proteins is not limited to plants, and could be earned out in any other eukaryotic cells, especially cells that can produce immunoglobulins such as mammalian cells.
The immunoglobulin chain sequences can be exchanged for sequences from other classes of immunoglobulins.
Other sequences can be incorporated into the construct in addition to the epitope tags and protease cleavage sites. For example, these could code for other functional regions, such as markers, enzyme activity, or sites for chemical coupling to other active molecules.
In addition to HIV gp120, two further examples have been developed. Similar constructs have been prepared using tetanus toxin non-toxic C-fragment (kindly donated by Dr. Neil Fairweather at Imperial College, London) and a tetanus toxin specific murine monoclonal antibody (kindly donated by Dr. Claus Koch at the State Serum Institute, Copenhagen). In addition, we have prepared corresponding constructs for the F-protein of bovine respiratory syncytial virus and a specific murine monoclonal antibody the genes for which were kindly provided by Dr. K.G. Madsen at the Danish Veterinary Institute for Virus Research.
Among the benefits of the invention, the following are noteworthy:-(i)Co-expression of antibody with antigen increases the levels of expression of antigen.
(ii)Antigen and whole antibody are genetically linked to form a type of immune complex. In other constructs, antigen and antibody fragments (that may or may not include the antigen binding site) are linked to form fusion proteins.
(iii)The recombinant antibody molecule and the antigen are automatically expressed at 1:1 ratio. In the case of constructs involving whole IgG, larger complexes may form spontaneously.
(iv)Antigen-antibody fusion molecules are easy to produce.
(v)The use of plants to make these molecules significantly reduces the cost, particularly if production is increased to agricultural scale.
(vi)For oral vaccines, consumption of edible transgenic plants offers an alternative, safe mode of delivery for immune complex vaccines.
(vii)There is potentially no need for addition of an adjuvant as the engineered molecule is already highly immunogenic. The CH2 and CH3 immunoglobulin domains include the site for complement activation and opsonisation of phagocytes Results:
Results have been obtained for two of the described constructs - CH2-gp120 and CH2-CH3-gp 120. RT-PCR confirmed that specific mRNA of the desired size was being made by transgenic plants (Figure 4).
Analysis by capture ELISA demonstrated that both constructs are being expressed by plants. We have gone on to characterise CH2-CH3-gp120 more fully and demonstrated that by ELISA that it contains IgG2a epitopes as well as gp120 epitopes as shown in Figures 5 and 6. Moreover, expression of the recombinant gp 120 is greater in plants expressing the CH2-CH3-gp120 construct, than those expressing either gp120 alone, gp 120 with a C-terminal HDEL tag, or CH2-gp 120. Preliminary results indicate an expression level of gp120 of approximately 0.8%.
Purification of plant derived material has been carried out and Western blot analysis demonstrates the presence of protein bands that are recognised by specific antibodies to gp120 and murine IgG2a (Figure 7). Under reducing conditions, the most prominent band has a molecular size consistent with that expected of approximately SOKd.
Under non-reducing conditions, higher molecular weight forms approaching 100Kd are detected. This is consistent with the formation of homodimers, through association of the CH2 and CH3 domains of the heavy chain.
A preliminary investigation of immunogenicity has been carned out in a single rhesus monkey. Following 2 administrations of purified plant derived CH2-CH3-gp 120 (150~g per dose) covalently linked with the adjuvant mycobacterial heat shock protein 70 using the SPDP bifunctional reagent, specific antibody and cellular immune responses were detected. After 3 immunisations, very significant T-cell proliferative responses were detected, not only to the plant CH2-CH3-gp120 construct, but also to CA 02388955 2002-04-24' purified recombinant gp120 expressed in Chinese hamster ovary cells. This indicates that the plant recombinant gp120 is correctly expressed and folded in an immunogenic conformation. Furthermore, four macaques have been immunised by the mucosal intra-vaginal route, and four by the targeted iliac lymph node route (Lehner et al., Nature Medicine, 2:767-775, 1996). After two immunisations, induction of interferon-y and interleukin-4 has been detected, indicating that both THl and TH2 responses are elicited. Further immunisation studies are being carned out in rhesus monkeys as well as mice.
Claims (64)
1. A method of increasing the production of a protein in cell comprising expressing recombinant DNA encoding a recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region.
2. The method of claim 1, wherein the desired protein is fused to the full length constant region of a heavy chain of an antibody.
3. The method of claim 1, wherein the desired protein is fused to a truncated constant region of a heavy chain of an antibody.
4. The method of claim 3, wherein the truncated region consists of one or more of the constant domains of a heavy chain of any antibody.
5. The method of claim 4, wherein the truncated region is from an IgG class antibody and consists of one or more of the CH1, CH2 and CH3 domains.
6. The method of any one of claims 1 to 5, wherein the desired protein is fused to the C-terminus of the constant region of a heavy chain of an antibody or to part of said constant region.
7. The method of any one of claims 1 to 5, wherein the desired protein is fused to the constant region of a heavy chain of an antibody or to part of said constant region, at a point within the constant region or within part of said constant region.
8. The method of any one of claims 1 to 7, wherein the desired protein is fused directly to said constant region or part thereof.
9. The method of any one of claims 1 to 7, wherein the desired protein is fused indirectly to said constant region or part thereof through a linking peptide.
10. The method of any one of the preceding claims, wherein the heavy chain is that of an antibody specific for the antigen.
11. The method of any one of the preceding claims, wherein the fusion protein contains a cleavage site for potential separation of the desired protein from the constant region or part thereof.
12. The method of any one of the preceding claims, wherein the fusion protein contains peptide tags.
13. The method of any one of the preceding claims, wherein the desired protein is a food protein or a therapeutic protein.
14. The method of claim 13, wherein the desired protein is an antigen.
15. The method of claim 14, wherein the antigen is human immnunodeficiency virus gp120.
16. The method of any one of the preceding claims, wherein the recombinant DNA
is expressed in eukaryotic host cells including those of a plant, fungus, insect or mammal.
is expressed in eukaryotic host cells including those of a plant, fungus, insect or mammal.
17.The method of any one of claim 16, wherein the recombinant DNA is expressed in a eukaryotic host cell which is capable of expressing an immunoglobulin.
18. The method of any one of claims 1 to 15, wherein the recombinant DNA is expressed in plant cells including those of dicotyledonous, monocotyledonous, leguminous or solanaceous plants.
19. The method of any one of the preceding claims, wherein the antibody heavy chain is human, rodent, rabbit, bovine, ovine, caprine, fowl, canine, camel, feline or primate.
20. A method of increasing the production of a protein in cell comprising expressing recombinant DNA encoding a recombinant fusion protein comprising a desired protein fused to the constant region of any member of the immunoglobulin supergene family or to part of said constant region.
21. A recombinant fusion protein comprising an antigen fused to the constant region of a heavy chain of an antibody or to a part of said constant region at a point within the constant region or within part of said constant region.
22.A recombinant fusion protein according to claim 21, in which the antigen is human immunodeficiency virus gp120.
23.A recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region, at a point within the constant region or within part of said constant region wherein the fusion protein does not comprise a functional antigen binding domain.
24. A fusion protein according to any one of claims 21 to 23, in which the antigen or desired protein is fused at a point within the full length constant region of the heavy chain of the antibody.
25. A fusion protein according to any one of claims 21 to 23, in which the antigen or desired protein is fused at a point within a truncated constant region of the heavy chain of the antibody.
26. A fusion protein according to claim 25, in which the truncated region is from an IgG class antibody and consists of one or more of the CH1, CH2 and CH3 domains.
27. A fusion protein according to any one of claims 21 to 26, in which the antigen or desired protein is fused directly to said constant region or part thereof.
28. A fusion protein according to any one of claims 21 to 26, in which the antigen or desired protein is fused indirectly to said constant region or part thereof through a linking peptide.
29. A fusion protein according to any one of claims 21 or 22, in which the heavy chain is that of an antibody specific for the antigen.
30. A fusion protein according to any one of claims 21 to 29, containing peptide tags.
31. A fusion protein according to any one of claims 21 to 30 in which the antibody heavy and light chains are human, rodent, rabbit, bovine, ovine, caprine, fowl, canine, camel, feline or primate.
32. A fusion protein according to any one of claims 21 to 31 in which the desired protein sequence is all or part of the HIV gp120 envelope protein linked to the combined heavy chain CH2-CH3 domain of an IgG class antibody.
33. A recombinant fusion protein comprising an antigen fused to the constant region of any member of the immunoglobulin supergene family or to part of the constant region at a point within the constant region or within part of said constant region.
34. A complex comprising two fusion proteins according to any one of claims 21 to 32 in parallel alignment.
35. A complex according to claim 34, also comprising antibody light chains which, together with the heavy chains of the fusion proteins or parts thereof, form the characteristic duplex symmetrical structure of an antibody or part thereof.
36. A fusion protein according to any one of claims 21 to 32 or a complex according to claim 34 or claim 35, produced in a plant or in a plant cell.
37. A vaccine comprising a recombinant fusion protein according to any one of claims 21 to 32 or a complex according to claim 34 or claim 35.
38. A method of forming a fusion protein according to any of claims 21 to 32 or a complex according to claim 34 or claim 35, which comprises expressing recombinant DNA coding for the components thereof in eukaryotic host cells including those of a plant, fungus, or mammal or a baculovirus.
39. A method of forming a fusion protein according to any of claims 21 to 32 or a complex according to claim 34 or claim 35, which comprises expressing recombinant DNA coding for the components thereof in eukaryotic cells which are capable of expressing an immunoglobulin.
40. A method of forming a fusion protein according to any of claims 21 to 32 or a complex according to claim 34 or claim 35, which comprises expressing recombinant DNA coding for the components thereof in plants that are dicotyledonous, monocotyledonous, leguminous or solanaceous.
41. A method of forming a complex according to claim 35, comprising expressing the heavy and light chains of the antibody or parts thereof in separate plants, isolating the heavy and light chains, and allowing the chains to associate to form the complex.
42. A method of forming a complex according to claim 35, comprising expressing the heavy and light chains of the antibody or parts thereof in a plant which expresses both the heavy and light chains.
43. The method of claim 44, wherein the plant which expresses both heavy and light chains is produced by crossing a plant which produces the heavy chain with a plant that produces the light chain.
44. A method according to claim 42, in which the host is a plant and the heavy and light chains of an antibody are co-expressed in the same plant.
45. A recombinant antibody molecule having an antigen fused to an antibody molecule, wherein the antibody molecule has affinity for the antigen.
46. The recombinant antibody molecule of claim 45, wherein the antigen is human immunodeficiency virus gp120.
47. The recombinant antibody molecule of claim 45 or claim 46, wherein the antibody molecule is complete monoclonal antibody or an antigen binding fragment thereof.
48. The recombinant antibody molecule of any one of claims 45 to 47, wherein the antibody molecule comprises a full length constant region of a heavy chain of an antibody and the antigen is fused to the full length constant region.
49. The recombinant antibody molecule of any one of claims 45 to 47, wherein the antibody molecule comprises a truncated constant region of a heavy chain of an antibody and the antigen is fused to a truncated constant region.
50. The recombinant antibody molecule according to claim 49, in which the truncated region is from an IgG class antibody and consists of one or more of the CH1, CH2 and CH3 domains.
51. The recombinant antibody molecule of any one of claims 48 to 50, wherein the antigen is fused to the C-terminus of the constant region or the part thereof.
52. The recombinant antibody molecule of any one of claims 48 to 50, wherein the antigen is fused to the constant region of a heavy chain of an antibody or to part of said constant region, at a point within the constant region or within part of said constant region.
53. The recombinant antibody molecule of any one of claims 45 to 52, wherein the antigen is fused directly to said antibody molecule.
54. The recombinant antibody molecule of any one of claims 45 to 52, wherein the antigen is fused indirectly to said antibody molecule through a linking peptide.
55. The recombinant antibody molecule of any one of claims 45 to 54, containing a cleavage site for potential separation of the antigen or desired protein from the antibody molecule.
56. The recombinant antibody molecule of any one of claims 45 to 55, wherein the antibody molecule comprises peptide tags.
57. The recombinant antibody molecule of any one of claims 45 to 56, wherein the antibody heavy and light chains are human, rodent, rabbit, bovine, ovine, caprine, fowl, canine, camel, feline or primate.
58. The recombinant antibody molecule of any one of claims 45 to 57, produced in a plant or in a plant cell.
59. An immune complex comprising a complex of the recombinant antibody molecules of any one of claims 45 to 58.
60. A vaccine comprising the recombinant antibody molecule of any one of claims 45 to 58 or the immune complex of claim 59.
61. A method of forming the recombinant antibody molecule of any one of claims 45 to 58, which comprises expressing recombinant DNA coding for the components thereof in prokaryotic host cells including those of a bacterium or archaebacteria.
62. A method of forming the recombinant antibody molecule of any one of claims 45 to 58, which comprises expressing recombinant DNA coding for the components thereof in eukaryotic host cells including these of a plant, fungus, or mammal or a baculovirus.
63. A method of forming the recombinant antibody molecule of any one of claims 45 to 58, which comprises expressing recombinant DNA coding for the components thereof in plants that are dicotyledonous, monocotyledonous, leguminous or solanaceous.
64. A transgenic plant expressing the a recombinant fusion protein according to any one of claims 21 to 32, or recombinant antibody molecule according to any one of claims 45 to 58.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9926084.6A GB9926084D0 (en) | 1999-11-03 | 1999-11-03 | Recombinant fusion molecules |
GB9926084.6 | 1999-11-03 | ||
PCT/GB2000/004216 WO2001032714A1 (en) | 1999-11-03 | 2000-11-03 | Recombinant fusion molecules |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2388955A1 true CA2388955A1 (en) | 2001-05-10 |
Family
ID=10863922
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002388955A Abandoned CA2388955A1 (en) | 1999-11-03 | 2000-11-03 | Recombinant fusion molecules |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP1226178A1 (en) |
JP (1) | JP2003512860A (en) |
CN (1) | CN1399646A (en) |
AU (1) | AU779719B2 (en) |
BR (1) | BR0015287A (en) |
CA (1) | CA2388955A1 (en) |
GB (1) | GB9926084D0 (en) |
HK (1) | HK1044956A1 (en) |
WO (1) | WO2001032714A1 (en) |
Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2475388A1 (en) * | 2002-02-14 | 2003-08-21 | William J. Rutter | Chimeric molecules for cleavage in a treated host |
US8029803B2 (en) | 2002-06-20 | 2011-10-04 | Paladin Labs, Inc. | Chimeric antigens for eliciting an immune response |
US8007805B2 (en) | 2003-08-08 | 2011-08-30 | Paladin Labs, Inc. | Chimeric antigens for breaking host tolerance to foreign antigens |
TW200526778A (en) * | 2003-11-14 | 2005-08-16 | Sembiosys Genetics Inc | Methods for the production of apolipoproteins in transgenic plants |
US20080305516A1 (en) * | 2004-11-30 | 2008-12-11 | Kristian Kjaergaard | Method of Producing Antibodies |
AU2006301846A1 (en) * | 2005-10-13 | 2007-04-19 | Akshaya Bio Inc. | Chimeric antigen containing hepatitis C virus polypeptide and FC fragment for eliciting an immune response |
US8444976B2 (en) | 2008-07-02 | 2013-05-21 | Argen-X B.V. | Antigen binding polypeptides |
GB2461546B (en) * | 2008-07-02 | 2010-07-07 | Argen X Bv | Antigen binding polypeptides |
EP3138854B1 (en) | 2009-03-10 | 2022-01-26 | Baylor Research Institute | Antibodies against cd40 |
US9562104B2 (en) | 2009-03-10 | 2017-02-07 | Baylor Research Institute | Anti-CD40 antibodies |
AU2010321676B2 (en) * | 2009-11-18 | 2015-02-26 | Agriculture Victoria Services Pty Ltd | Angiogenin expression in plants |
AU2015202777B2 (en) * | 2009-11-18 | 2017-09-14 | Agriculture Victoria Services Pty Ltd | Angiogenin expression in plants (2) |
JP7037884B2 (en) | 2014-01-13 | 2022-03-17 | ベイラー リサーチ インスティテュート | New vaccines for HPV and HPV-related diseases |
WO2015190885A1 (en) * | 2014-06-12 | 2015-12-17 | 중앙대학교 산학협력단 | Method for manufacturing transgenic plant producing immunogenic complex proteins and immunogenic complex proteins obtained therefrom |
US10610585B2 (en) | 2017-09-26 | 2020-04-07 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Methods and compositions for treating and preventing HIV |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU660297B2 (en) * | 1990-11-09 | 1995-06-22 | Stephen D. Gillies | Cytokine immunoconjugates |
WO1998036087A1 (en) * | 1997-02-13 | 1998-08-20 | American National Red Cross | Immunological tolerance to hiv epitopes |
AU758240B2 (en) * | 1998-02-25 | 2003-03-20 | Merck Patent Gmbh | Enhancing the circulating half-life of antibody-based fusion proteins |
-
1999
- 1999-11-03 GB GBGB9926084.6A patent/GB9926084D0/en not_active Ceased
-
2000
- 2000-11-03 WO PCT/GB2000/004216 patent/WO2001032714A1/en not_active Application Discontinuation
- 2000-11-03 BR BR0015287-0A patent/BR0015287A/en not_active IP Right Cessation
- 2000-11-03 EP EP00973028A patent/EP1226178A1/en not_active Withdrawn
- 2000-11-03 CA CA002388955A patent/CA2388955A1/en not_active Abandoned
- 2000-11-03 CN CN00814715A patent/CN1399646A/en active Pending
- 2000-11-03 JP JP2001535412A patent/JP2003512860A/en active Pending
- 2000-11-03 AU AU11584/01A patent/AU779719B2/en not_active Ceased
-
2002
- 2002-08-29 HK HK02106395.6A patent/HK1044956A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
HK1044956A1 (en) | 2002-11-08 |
BR0015287A (en) | 2002-07-09 |
WO2001032714A1 (en) | 2001-05-10 |
EP1226178A1 (en) | 2002-07-31 |
AU1158401A (en) | 2001-05-14 |
JP2003512860A (en) | 2003-04-08 |
GB9926084D0 (en) | 2000-01-12 |
AU779719B2 (en) | 2005-02-10 |
CN1399646A (en) | 2003-02-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Stöger et al. | Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies | |
AU779719B2 (en) | Recombinant fusion molecules | |
EP0807173B1 (en) | Methods for producing immunoglobulins containing protection proteins in plants and their use | |
Paul et al. | Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV | |
WO1996021012A9 (en) | Methods for producing immunoglobulins containing protection proteins in plants and their use | |
US20080115244A1 (en) | Method for producing immunoglobulins containing protection proteins and their use | |
JP2008515454A (en) | System and method for antibody production in plant cell culture | |
TW200907059A (en) | Recombinant expression vector elements (rEVEs) for enhancing expression of recombinant proteins in host cells | |
US20030051275A1 (en) | Methods and means for expression of mammalian polypeptides in monocotyledonous plants | |
JP2003531623A (en) | Immunoglobulin binding protein array in plant cells | |
KR102453605B1 (en) | Novel Peptide Tag, Antibodies Binding to the Same and Uses thereof | |
US8557246B2 (en) | Fusion protein that directs vaccine antigens to antigen-presenting cells, and applications thereof | |
AU729592B2 (en) | Secretory immunoglobulin A as a mucosal vaccine delivery system | |
CA2019323A1 (en) | Chimeric mouse-human km10 antibody with specificity to a human tumor cell antigen | |
US20060018900A1 (en) | Self antigen vaccines for treating B-cell lymphomas and other cancers | |
CN116003611A (en) | anti-TMPRSS 2 antibodies and uses thereof | |
KR100923664B1 (en) | A vector for cell surface expression of pig IgG Fc domian, a host cell transformed with the vector, a vaccine against viruses related to pig diseases and a manufacturing method of the same using the host cell | |
CN113603790A (en) | RGD-anti-p 21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof | |
AU2010288743A1 (en) | Antibody fusion-mediated plant resistance against Oomycota | |
JPH02128698A (en) | Method for producing in vivo chimera antibody by locus specific recombination | |
US20050138692A1 (en) | Production of cancer-specific antibodies in plants | |
AU773602B2 (en) | Methods for producing immunoglobulins containing protection proteins in plants and their use | |
CN117285633A (en) | Pig RIG-I like receptor LGP2 specific monoclonal antibody and application | |
Edgü | Malaria immunoassemblins: A novel combinatorial vaccine approach against plasmodium falciparum based on highly improved Fc-fusion proteins | |
KR20020059413A (en) | Creation of variable length and sequence linker regions for dual-domain or multi-domain molecules |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Discontinued |