CN1087681A - New antibodies with passive immunity of pathogenic bacterial infection in the anti-human body - Google Patents
New antibodies with passive immunity of pathogenic bacterial infection in the anti-human body Download PDFInfo
- Publication number
- CN1087681A CN1087681A CN93119080A CN93119080A CN1087681A CN 1087681 A CN1087681 A CN 1087681A CN 93119080 A CN93119080 A CN 93119080A CN 93119080 A CN93119080 A CN 93119080A CN 1087681 A CN1087681 A CN 1087681A
- Authority
- CN
- China
- Prior art keywords
- antibody
- sequence
- light chain
- heavy chain
- variable
- 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.)
- Pending
Links
- 230000001717 pathogenic effect Effects 0.000 title description 21
- 230000036039 immunity Effects 0.000 title description 11
- 208000035143 Bacterial infection Diseases 0.000 title description 2
- 208000022362 bacterial infectious disease Diseases 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 60
- 238000000034 method Methods 0.000 claims abstract description 56
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 48
- 239000000203 mixture Substances 0.000 claims abstract description 28
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 25
- 241000223960 Plasmodium falciparum Species 0.000 claims abstract description 24
- 239000002773 nucleotide Substances 0.000 claims description 65
- 125000003729 nucleotide group Chemical group 0.000 claims description 65
- 230000004927 fusion Effects 0.000 claims description 61
- 101100328487 Arabidopsis thaliana NFS2 gene Proteins 0.000 claims description 60
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 48
- 235000018102 proteins Nutrition 0.000 claims description 46
- 239000012634 fragment Substances 0.000 claims description 44
- 210000004027 cell Anatomy 0.000 claims description 42
- 239000000427 antigen Substances 0.000 claims description 34
- 102000036639 antigens Human genes 0.000 claims description 34
- 108091007433 antigens Proteins 0.000 claims description 33
- 241000224016 Plasmodium Species 0.000 claims description 31
- 238000002360 preparation method Methods 0.000 claims description 27
- 230000000890 antigenic effect Effects 0.000 claims description 26
- 108060003951 Immunoglobulin Proteins 0.000 claims description 24
- 102000018358 immunoglobulin Human genes 0.000 claims description 24
- 239000013612 plasmid Substances 0.000 claims description 24
- 239000000463 material Substances 0.000 claims description 18
- 230000000078 anti-malarial effect Effects 0.000 claims description 13
- 239000003430 antimalarial agent Substances 0.000 claims description 13
- 208000015181 infectious disease Diseases 0.000 claims description 13
- 230000008859 change Effects 0.000 claims description 12
- 230000000295 complement effect Effects 0.000 claims description 12
- 238000001890 transfection Methods 0.000 claims description 9
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 8
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 8
- 210000004962 mammalian cell Anatomy 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 241000124008 Mammalia Species 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 4
- JTXVXGXTRXMOFJ-FXQIFTODSA-N Asn-Pro-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O JTXVXGXTRXMOFJ-FXQIFTODSA-N 0.000 claims description 3
- UVKNEILZSJMKSR-FXQIFTODSA-N Pro-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H]1CCCN1 UVKNEILZSJMKSR-FXQIFTODSA-N 0.000 claims description 3
- NALWOULWGHTVDA-UWVGGRQHSA-N Asp-Tyr Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NALWOULWGHTVDA-UWVGGRQHSA-N 0.000 claims 2
- 108010068265 aspartyltyrosine Proteins 0.000 claims 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 239000000539 dimer Substances 0.000 claims 1
- 239000013600 plasmid vector Substances 0.000 claims 1
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 abstract description 56
- 201000004792 malaria Diseases 0.000 abstract description 17
- 238000011282 treatment Methods 0.000 abstract description 8
- 208000030852 Parasitic disease Diseases 0.000 abstract description 3
- 230000003053 immunization Effects 0.000 abstract description 2
- 235000001014 amino acid Nutrition 0.000 description 84
- 150000001413 amino acids Chemical class 0.000 description 84
- 150000007523 nucleic acids Chemical group 0.000 description 54
- 108020004414 DNA Proteins 0.000 description 52
- 108020004707 nucleic acids Proteins 0.000 description 51
- 102000039446 nucleic acids Human genes 0.000 description 51
- 239000003795 chemical substances by application Substances 0.000 description 29
- 238000001514 detection method Methods 0.000 description 19
- 238000005516 engineering process Methods 0.000 description 16
- 210000003046 sporozoite Anatomy 0.000 description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 239000013604 expression vector Substances 0.000 description 11
- 230000004048 modification Effects 0.000 description 11
- 238000012986 modification Methods 0.000 description 11
- 102000005962 receptors Human genes 0.000 description 11
- 108020003175 receptors Proteins 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 10
- 201000010099 disease Diseases 0.000 description 10
- 239000012636 effector Substances 0.000 description 10
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 210000004408 hybridoma Anatomy 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 230000003449 preventive effect Effects 0.000 description 9
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000010353 genetic engineering Methods 0.000 description 7
- 230000008521 reorganization Effects 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 241001529936 Murinae Species 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 230000009545 invasion Effects 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 101000726057 Plasmodium falciparum Circumsporozoite protein Proteins 0.000 description 5
- 108020004511 Recombinant DNA Proteins 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- UKBSDLHIKIXJKH-HJGDQZAQSA-N Thr-Arg-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O UKBSDLHIKIXJKH-HJGDQZAQSA-N 0.000 description 4
- AVYVKJMBNLPWRX-WFBYXXMGSA-N Trp-Ala-Ser Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 AVYVKJMBNLPWRX-WFBYXXMGSA-N 0.000 description 4
- 244000309464 bull Species 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 244000045947 parasite Species 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 210000004215 spore Anatomy 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 101100080609 Drosophila melanogaster Nsf2 gene Proteins 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 241001505293 Plasmodium ovale Species 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000013599 cloning vector Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- -1 polyethylene Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 230000003252 repetitive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 102000006734 Beta-Globulins Human genes 0.000 description 2
- 108010087504 Beta-Globulins Proteins 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- LOJYQMFIIJVETK-WDSKDSINSA-N Gln-Gln Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(O)=O LOJYQMFIIJVETK-WDSKDSINSA-N 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 241000223821 Plasmodium malariae Species 0.000 description 2
- 241000223810 Plasmodium vivax Species 0.000 description 2
- PIQRHJQWEPWFJG-UWJYBYFXSA-N Ser-Tyr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PIQRHJQWEPWFJG-UWJYBYFXSA-N 0.000 description 2
- AUZADXNWQMBZOO-JYJNAYRXSA-N Tyr-Pro-Arg Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CC=C(O)C=C1 AUZADXNWQMBZOO-JYJNAYRXSA-N 0.000 description 2
- AGDDLOQMXUQPDY-BZSNNMDCSA-N Tyr-Tyr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O AGDDLOQMXUQPDY-BZSNNMDCSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000002223 anti-pathogen Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000012050 conventional carrier Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 1
- BHNQPLPANNDEGL-UHFFFAOYSA-N 2-(4-octylphenoxy)ethanol Chemical compound CCCCCCCCC1=CC=C(OCCO)C=C1 BHNQPLPANNDEGL-UHFFFAOYSA-N 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
- FQYRLEXKXQRZDH-UHFFFAOYSA-N 4-aminoquinoline Chemical compound C1=CC=C2C(N)=CC=NC2=C1 FQYRLEXKXQRZDH-UHFFFAOYSA-N 0.000 description 1
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 101150052200 CS gene Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000002476 Falciparum Malaria Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102100036263 Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial Human genes 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001001786 Homo sapiens Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial Proteins 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 101900205473 Plasmodium falciparum Circumsporozoite protein Proteins 0.000 description 1
- 201000011336 Plasmodium falciparum malaria Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710194807 Protective antigen Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 101710152205 Sporozoite antigen Proteins 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 229960003677 chloroquine Drugs 0.000 description 1
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002650 habitual effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229940124452 immunizing agent Drugs 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 150000002678 macrocyclic compounds Chemical class 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000036281 parasite infection Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 238000013326 plasmid cotransfection Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
- C07K16/205—Plasmodium
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
This paper provides the protein and the peptide of the monoclonal antibody that is derived from the mouse plasmodium falciparum, comprise the variable light chain of synthetic hommization and the variable heavy chain sequence that are used for the treatment of in the method and composition, the CDR peptide, and hommization antibody, described composition can provide passivity immunizing power to the parasitic infection that causes malaria.
Description
Roughlly speaking, for example the present invention relates at selected pathogenic agent, plasmodium, on the mono-clonal and the recombinant antibodies of epitope.Composition and preparation and these method for compositions of use of containing these antibody.
Malaria is a kind of serious, the wide-scale distribution disease, and it is that each kind by the protozoon parasite plasmodium causes, comprises and infects four human kinds.For example, plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and malariae, Plasmodium ovale and malariae [referring to, for example, V.Enea et al., Science, 225:628-630(1984)].Owing to do not control the effective vaccine and the plan of vector colony and new drug resistance strain, thereby malaria is still to be present in the world the most extensive today and to cause one of disease.Roughlly speaking, the treatment of malaria mainly relies on preventive medicine, as the 4-quinolylamine.But, for most of cases, the resistance that plasmodium falciparum causes and produce the effect that some undesirable side effects has been damaged these rems.
In the malaria disease prevention area more research project concentrate on plasmodium parasites sporozoite form, particularly ring spore (CS) protein [Clyde et al., Am.J.Trop.Med.Hyg., 24:397(1975); Rieckman et al., Bull.WHO, 57(1): 261(1979); And U.S.Patent4,957,869].Existing people has reported the CS protein gene of many kinds in the plasmodium or its segmental cloning and feature and recombinant expressed in intestinal bacteria or yeast host cell thereof.The proteinic central iteron of CS is immunodominant, that is, if the technician injects animal with sporozoite, this animal produces the antibody of anti-iteron.First kind of anti-sporozoite candidate vaccine testing at human body is to be foundation with the repetition epitope that exists on plasmodium falciparum CS protein, and this epitope is by (AsnAlaAsnPro)
37(AsnValAspPro)
4Form this epitope not variation all the time in many bacterial strains of being checked up to now.A kind of candidate vaccine that utilization is called R3 2 tet3 2 carries out clinical trial, produces the protective response that anti-sporozoite excites in the people of aspiration, wherein vaccine by
NH
2-Met-Asp-Pro-[（Asn-Ala-Asn-Pro）
15（Asn-Val-Asp-Pro）
1]
2-Leu-Arg-Arg-Thr-His-Arg-Gly-Arg-His-His-Arg-Arg-His-Arg-Cys-Gly-Cys-Trp-Arg-Leu-Tyr-Arg-Arg-His-His-Arg-Trp-Gly-Arg-Ser-Gly-Ser-COOH，
Form [referring to, Ballou et al., The Lancet, June 6,1987, pp.1277-1281; And European Patent Publication No.0192626, on August 27th, 1986 published, the document is incorporated herein by reference].
Differentiated that many it is effective [Y.Charoenvit et al. at proof in proteinic many monoclonal antibodies (mAbs) that derive from each stage of plasmodium life cycle and the passive transfer test carried out in mouse and monkey, J.Immunol., 146(3): 1020-1025(1990)].However, use Antybody therapy or prevention of malaria to still have shortcoming.Because the unfavorable immunne response that human body produces at exogenous antibodies for example, is removed and toxic side effect fast, the antibody that has limited mouse or other animals is applied to human body.Verified is at the immunoglobulin (Ig) constant region of murine antibody and variable region in the intravital above-mentioned immunne response of people.
Several technology have been described in the prior art, suggestion changes the antibody of mouse (with other kinds) to be reduced in desired species, for example occur among the mankind at parental antibody immunne response [referring to, for example, PCT patent application No.PCT/WO86/01533, on March 13rd, 1986 published; UK Patent Application No.GB2188638A, on October 7th, 1987 published; People such as Amit, Science, 233:747-753(1986); People such as Queen, Proc, Natl.Acad.Sci.USA, 86:10029-10033(1989); PCT patent application No.PCT/WO90/07861, publish July 26 nineteen ninety; And Riechmann et al., Nature, 332:323-327(1988)].Although prior art has been advised possible experimental technique, do not point out how to provide for growth in the body of effective inhibition plasmodium falciparum required in conjunction with feature.
In this area, still need another kind that the method for the immunity of anti-selected pathogenic agent such as malarial parasite infection is provided, the preventive that can provide effective short-term to protect especially is provided.
On the one hand, the invention provides complementary determining region (CDR) peptide that comes from the monoclonal antibody of the selected epitope on a kind of pathogenic agent, and the fragment of these peptides and analogue.Preferably, this antibody capable is in conjunction with a plasmodial epitope, particularly CS iteron epitope or its fragment, for example mouse-anti plasmodium falciparum mAb NFS2.These CDRs have kept the antigen-binding specificity of the mAb that obtains from its source.
On the other hand, the invention provides a kind of stripped, natural existence or synthetic, the immunoglobulin light of hommization or the aminoacid sequence of variable region of heavy chain, this sequence comprises that one or more come from a kind of CDR sequence of light or heavy chain of selected antibody.
Also have an aspect, the invention provides a kind of containing to come from a kind of antimalarial protozoon antibody, a kind of antimalarial protozoon CDR, a kind of fusion rotein of the variable light chain of its a kind of function fragment or analogue and/or first aminoacid sequence of heavy chain.The aminoacid sequence that this is selected is by connecting after operating or being fused on second selected aminoacid sequence.These fusion roteins have the antigen-binding specificity of mAb, and the first above-mentioned selected aminoacid sequence derives from this mAb.
One side more of the present invention provides a kind of to selected plasmodium antigens determinant, and for example, the plasmodium falciparum repeat region has specific genetic engineering antibody.
On the other hand, the invention provides a kind of plasmodium falciparum antibody or its fragment that produces by screening hybridoma product, this hybridoma rule of origin is in the whole components of any kind of immunoglobulin (Ig) with mAb NFS2 antigenic determinant, or people or murine antibody combinatorial library.
Another aspect the invention provides above-described Fab fragment of passing through antibody or anti--plasmodium mAbs of processing.
As another additional aspect, the invention provides coding protein described herein, peptide, antibody and segmental nucleotide sequence, and the plasmid that contains one or more these nucleotide sequences, use its host transformed, and, for example produce the method for these nucleotide sequence expression products in the mammalian cell at host cell.
Other aspects provided by the invention comprise provides a kind of pharmaceutical composition and a kind of prevention method of passive immunity can for the human body that might contact malarial parasite, said composition contains a kind of protein at least a described herein of significant quantity, antibody, acceptable carrier or thinner on peptide or fragment and a kind of pharmacology.
Describe in the preferred embodiment that other aspects of the present invention and advantage further are discussed in more detail below.
Fig. 1 describes the amino acid and the nucleotide sequence of the naturally occurring variable region of light chain of mAb NFS2.
Fig. 2 describes amino acid and the nucleotide sequence of the synthetic humanized light chain variable district pfhzlcl-1 that contains anti--plasmodium CDRs.The part of beneath line is CDRs.
Fig. 3 has described amino acid and the nucleotide sequence of synthetic humanized light chain variable district pfhzlcl-2.
Fig. 4 has described the amino acid and the nucleotide sequence of the variable region of heavy chain of naturally occurring mAb NFS2.
Fig. 5 has described amino acid and the nucleotide sequence of a kind of synthetic hommization variable region of heavy chain pfhzhc2-4.
Fig. 6 has described amino acid and the nucleotide sequence of synthetic hommization variable region of heavy chain pfhzhc2-3.
Fig. 7 graphic representation is used to express the pfhzhc2-3-Pcd plasmid of synthetic antimalarial protozoon heavy chain in mammalian cell.This plasmid contains a kind of β-Nei Xiananmei (gene of β-lac), a kind of SV40 copy source (SV40), a kind of cytomegalovirus promoter sequence (CMV), synthetic heavy chain pfhzhc2-3, derive from the poly a-signal of Trobest (BGH), a kind of beta Globulin promotor (β glopro), a kind of dehydrofolic acid reductase gene (DHFR), and from the another kind of BGH sequence poly a-signal of background pUC19.
Fig. 8 is the pfhzlcl-1-Pcn plasmid figure that graphic representation is used for expressing at mammalian cell synthetic light chain.This plasmid figure is different from the plasmid among Fig. 7, and its difference is that this plasmid contains synthetic humanized light chain pfhzlcl-1, rather than its heavy chain, and replaces DHFR with a neomycin gene (Neo).
Fig. 9 has described Nucleotide and the aminoacid sequence of synthetic hommization variable region of heavy chain pfhzhc2-6.
The invention provides and can make the people who is inoculated have an anti-short time length by selected pathogenic infection people, the preventive of protective immunity state for example, is used to the patient who controls infection and be used for having contacted this pathogenic agent.Reorganization or through the antibody of processing, preferably chimeric, hommization or human monoclonal antibodies can be used as such passive protection albumen.Prevention these protein in the composition can with before pathogenic agent contact for medicine, and do not require and take successive doses every day to mediate short-term protection.
Though following description specifically refers to provide the antibody to the passivity prophylactic effect of the sporozoite form of pathogenic agent-plasmodium falciparum (a kind of paathogenic factor of human malaria), but the invention of Miao Shuing has more than the pathogenic agent that is limited to any particular state herein, neither be only limited to a kind of independent pathogenic agent.In accordance with the teachings of the present invention, those of skill in the art can make up at selected pathogenic agent in this area, and for example, other kind plasmodiums comprise the blood phase, liver phase, or other recombinant antibodies of gamont phase.Can also make up at other people infectious parasite according to the present invention, for example, malariae, the antibody of the present invention of the ring spore CS gene of Plasmodium vivax and Plasmodium ovale is to be provided for the passive transfer protein of anti-these parasitic infections.Equally, can relate to other infectious agent, virus, bacterium or the like according to passive therapeutical agent provided by the invention.In addition, also can be with the therapeutical agent of such antibody as the treatment acute infection period.
1. definition
" first merges the pairing body " is meant a nucleotide sequence of encoding amino acid sequence, this aminoacid sequence can be a heavy chain immunoglobulin whole or part, a light chain, and contain from the variable region of one or two chain with and function fragment or a kind of analogue of CDRs.They have selected high-titer antibody, murine antibody preferably, the antigen-binding specificity of NFS2.
" second merges the pairing body " is meant the nucleotide sequence of a kind of protein of coding or peptide, and the first fusion pairing body and this nucleotide sequence carry out framework and merge, and perhaps merge for helping a kind of optional conventional joint sequence.Situation is that this second fusion pairing body and the first fusion pairing body are allogenic preferably.Second merges the pairing body can comprise second meaningful antibody district of coding, for example, and whole or the suitable people variable region of part or the nucleotide sequence in skeleton district.
" fusion molecule " is meant that first merges the pairing body by being connected to second product that merges on the pairing body after operating." can be operatively connected " of merging the pairing body is defined as the anti-plasmodium falciparum sequence (first merges the pairing body) that can make from the antigen-specific of donor antibody and has second of required feature merges a kind of combination that the pairing body surface reaches.For example, can use or not use a kind of nucleotide sequence of coded amino acid joint, perhaps be connected to second and merge on the pairing body by the framework fusion.
" fusion rotein " is meant by fusion molecule encoded protein matter, can obtains this protein by fusion molecule is expressed in selected host cell.This fusion rotein is through finished antibody, for example, and chimeric or human nature antibody, or merge any antibody fragment to immunoglobulin (Ig) or the NIg or the like in the present invention's identification.
" donor antibody " is meant its natural existence or modified lightening and/or heavy chain, its variable region, its CDRs or other function fragments merge the pairing body as first so that provide fusion molecule with donor antibody antigen-specific and the antibody (polyclone, mono-clonal or reorganization) of fusion rotein.Be applicable to that in the present invention the donor antibody of using is mouse mAb NFS2 and other antibody of describing hereinafter.
" receptor antibody " is meant a kind of and donor antibody allos, but with the patient that will treat (people or other Mammalss) homologous antibody (polyclone, mono-clonal or reorganization), the sequence of the whole or any part of this antibody its variable heavy and/or light chain skeleton district and/or its is heavy and/or light chain constant region is used as second and merges the pairing body.Preferred situation is as receptor antibody with people's antibody.
The definition of " CDRs " is the complementary determining region aminoacid sequence of antibody, and it is the hypervariable region of heavy and light chain.CDRs is provided for antibody and antigen or the main contact residues of antigenic determinant bonded.The significant CDRs of the present invention derives from the variable heavy and sequence of light chain of donor antibody, and comprises function fragment and the analogue of naturally occurring CDRs, and it has or kept the antigen-binding specificity of the donor antibody that is same as its source.
" total antigen-binding specificity " is meant, for example, though mAb NFS2 has the antigen affinity feature of certain level, and the CDR by the nucleic acid sequence encoding of NFS2 in suitable structural environment has lower affinity, but the CDRs that is desirably in NFS2 in the above-mentioned environment is same as identification the antigenic determinant of NFS2.
" function fragment " is to have kept the source that is same as to give birth to the portion C DR sequence of this segmental antibody antigen binding specificity or partly heavy or light chain variable sequence.
" analogue " be meant by replacing at least a amino acid, changes or chemistry replaces a seed amino acid and modified amino acid or peptide sequence, and this modification makes aminoacid sequence keep the biological property of unmodified sequence, for example, and antigen-specific.
" allelic variation or modification " is the variation of carrying out in the nucleotide sequence of code book invention amino acid or peptide sequence.Above-mentioned variation or modification be owing to the genetic code degeneracy causes, or provide required feature through after the special processing.These variations or modification cause or do not cause the variation of coded aminoacid sequence.
" engineered antibody " is a kind of fusion rotein type, be a kind of synthetic antibody (for example, a kind of chimeric or hommization antibody), wherein by coming from the part that the CDRs similar portions that has at specific one or more donor antibody of selected antigenic determinant has substituted the light and/or variable region of heavy chain on a kind of selected receptor antibody.These engineered antibodies also have change the light and/or variable region of heavy chain skeleton zone of coding receptor antibody nucleotide sequence to keep the feature of donor antibody binding specificity.These antibody can comprise immunoglobulin (Ig) constant region and the variable skeleton district that comes from receptor antibody, and one or more CDRs that come from plasmodium donor antibody described herein.Preferred engineered antibody is to adopt the recombinant DNA technology preparation among the present invention.
" chimeric antibody " is meant a type of engineered antibody, and it contains naturally occurring variable region light chain and the heavy chain (CDR and skeleton district) that derives from inhuman donor mAb and derives from the binding substances of the light and heavy chain constant region of a kind of people (or other allos animals) acceptor mAb.
" hommization antibody " is meant a kind of engineered antibody, and it has CDRs and/or other parts in the light and/or heavy chain variable domain skeleton district that derives from inhuman donor immunity sphaeroprotein, and rest part is the part molecule that comes from one or more human normal immunoglobulins.Such antibody also comprises and it is characterized by the hommization heavy chain that combines with unmodified light chain or a kind of chimeric light chain of a kind of donor or acceptor or the engineered antibody of reverse situation.
" effector agent " is meant the nonprotein carrier molecule, adopts ordinary method to combine fusion rotein on this molecule, and/or other fragments of natural or synthetic light or heavy chain or this donor antibody of this donor antibody.Such nonprotein carrier can be included in the conventional carrier that uses in the diagnostic field, for example, and polyethylene or other plastic beads, or other non-proteinaceous matter of in medical field, using and can give the humans and animals administration safely.Other effector agents also comprise and are used for a kind of heavy metal atom of chelating, or as the macrocycle molecule of the toxin of ricin class.Such effector agent can be used for increasing the transformation period of antimalarial protozoon deutero-aminoacid sequence or being used to increase its characteristic.
II. antimalarial protozoon antibody
In order to make up the recombinant antibodies relevant of the present invention, can use inhuman kind coming from the presence of the plasmodium strain antigens that can infect human body to produce gratifying donor antibody with the pathogenic agent that causes malaria.Adopt conventional hybridoma technology can secrete the hybridoma cell line of anti-selected antigenic inhuman mAb with preparation.As an embodiment, identified mouse mAb, NFS2 is needed antibody, this antibody can be used for preparing chimeric or hommization antibody of the present invention.
Mouse IgG mAb NFS2h has the antigen-binding specificity at plasmodium falciparum CS albumen iteron.In vitro detection, it can hinder sporozoite and invade human liver cell or liver cancer cell.Similar antibody has made mouse have antimalarial passivity provide protection in the mouse model animal.Described the preparation process of mAb NFS2 in the following Example 1 in detail.
The invention is not restricted to for example NFS2mAb with and the use of alterable height sequence.In the following description, the donor mAb note is made NFS2, the purpose that designs such scheme only is to oversimplify in order to make to describe.Can be with other antimalarial protozoon antibody surrogates.Suitable antibody comprises, the mouse mAb2A10 of for example anti-CS repetitive proteins, or at R.A.Wirtz et al., Bull WHO, 65:39-45(1987) middle other mAbs that describe.
Can use equally in the present invention antibody that other animals of being protected by sporozoite or protective antigen determinant immunization with selected plasmodium kind produce as protectiveness anti--source of plasmodium sequence.
For example, can use plasmodium falciparum CS protein iteron protein R32tet32
NH
2-Met-Asp-Pro－[（Asn-Ala-Asn-Pro）
15（Asn-Val-Asp-Pro）
1]
2-Leu-Arg-Arg-Thr-His-Arg-Gly-Arg-His-His-Arg-Arg-His-Arg-Cys-Gly-Cys-Trp-Arg-Leu-Tyr-Arg-Arg-His-His-Arg-Trp-Gly-Arg-Ser-Gly-Ser-COOH
Induce people and mouse mAbs with binding specificity.This iteron albumen is the suitable target that is used for screening the neutrality antibody that can use at the preventive that anti-malarial infects.
Equally, the antigenic determinant that NFS2 can be able to be replied with and analogue be used to screen and prepare other antimalarial protozoon antibody, can protect the people to resist the malaria prophylaxis composition in a short time to be used for preparation.Compelling other antigenic determinants comprise the non-repeatability lateral areas antigenic determinant of plasmodium kind, other duplicate domains or various liver and the antigenic determinant of blood and sexual phase.Understanding to these antigenic determinants can make arbitrary technician's synthetic and the naturally occurring passive or active immunity peptide that is suitable for providing anti-plasmodium falciparum or other plasmodium kinds of identification in this area.It is human that these knowledge also make the mAbs of production can be used for the prevention of malaria infection.
For example, utilize the mouse mAb antigenic determinant of describing in this article by screening hybridoma or other associating libraries, or antibody phage display storehouse [W.D.Huse et al., Science, 246:1275-1281(1988)] and prepare other plasmodium falciparum antibody.Compete detection method with routine,, use one or more antigenic determinants of describing to screen herein and comprise the hybridoma product, or come from the full component antibody of any kind of immunoglobulin (Ig) at an interior antibody aggregate as describing among the embodiment hereinafter.
As those antibody of describing hereinbefore, comprise that those include but not limited at the antibody of a kind of required antigenic determinant and with ordinary method, coding mouse mAbs, people mAbs, the antibody that produces of gene, and association's antibody all can be used for as donor antibody, as the antibody fragment source, and the prevention composition that is used for the plasmodium falciparum of anti-human body.Preferably, reply plasmodium, especially plasmodium falciparum, the antibody that produces during antigenic determinant can be used as at the preparation fusion rotein, comprise the variable heavy and/or light-chain amino acid sequence that needs in the genetic engineering antibody, or its function fragment (for example, donor CDRs).Therefore, the present invention also can utilize the donor antibody except that NFS2, these antibody capables be attached to basically by the aminoacid sequence of repetitive proteins with and the plasmodium falciparum peptide formed of analogue on.
In addition, can further change or operate in herein the mAbs that differentiates, other use sporozoite, that R32tet32 or the repetition antigenic determinant of differentiating herein prepare and to its mAbs that replys to make it to have other required prevention characteristic.
III. antibody fragment, amino acid and nucleotide sequence
The invention provides variable light chain and variable heavy chain sequence that stripped naturally occurring or synthetic comes from mAb NFS2, and CDRs and its fragment, these compositions all can be used for designing the fusion rotein (comprising genetic engineering antibody) with this mAb antigen-binding specificity.
The variable heavy chain of naturally occurring NFS2 has the amino acid described and the feature of nucleotide coding sequence in Fig. 4.This chain has the feature of the CDRs of the amino acid whose sequence that contains following Nucleotide and supposition.CDR1 has following sequence signature:
AGCTATGCCATGTCT
SerTyrAlaMetSer.
Naturally occurring CDR2 nucleic acid and aminoacid sequence are:
GAAATTAGTGATGGTGGTAGTTACACCTACTATCCAGACACTGTGACGGGC
GluIleSerAspGlyGlySerTyrThrTyrTyrProAspThrValThrGly.
Naturally occurring CDR3 has nucleic acid and aminoacid sequence is:
CTCATCTACTATGGTTACGACGGGTATGCTATGGACTAC
LeuIleTyrTyrGlyTyrAspGlyTyrAlaMetAspTyr.
The NFS2 variable region heavy chain of synthetic human nature has the amino acid that shows and the feature of nucleic acid coding sequence in Fig. 5 and Fig. 6.In two synthetic chains, CDRs has the aminoacid sequence of following Nucleotide and supposition.Prepare the CDR1 that Nucleotide changes by naturally occurring CDRs.And the variation of Nucleotide is represented in line place down below.Synthetic CDR1 has following sequence signature:
AGCTATGCCATGAGC
SerTyrAlaMetSer.
Synthetic CDR2 nucleic acid and aminoacid sequence are same as naturally occurring sequence.Synthetic CDR3 has nucleic acid and the aminoacid sequence that is same as naturally occurring CDR3.
The variable light chain of naturally occurring NFS2 has the feature of aminoacid sequence and nucleic acid coding sequence shown in Figure 1.The feature that this chain also has is the CDRs that following Nucleotide and aminoacid sequence are arranged.Nucleic acid and aminoacid sequence that CDR1 has are characterized as:
AAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAATTACTTGGCC
LysSerSerGlnSerLeuLeuTyrSerSerAsnGlnLysAsnTyrLeuAla.
Nucleic acid and aminoacid sequence that CDR2 has are characterized as:
TGGGCATCCACTAGGGAATCT
TrpAlaSerThrArgGluSer.
Nucleic acid and aminoacid sequence that CDR3 has are characterized as:
CAGCAATATTATAGCTATCCTCGGACG
GlnGlnTyrTyrSerTyrProArgThr.
The variable light chain of the NFS2 of synthetic hommization has the amino acid that shows among Fig. 2 and the feature of nucleic acid coding sequence.This chain has the aminoacid sequence that contains following supposition and by shown nucleotide sequence coded CDRs feature.
Prepare three CDRs that Nucleotide changes by naturally occurring corresponding CDRs.Beneath line part is represented this change.Nucleic acid and aminoacid sequence that synthetic CDR1 has are characterized as:
AAGAGCTCTCAGAGCCTTTTATACTCGAGCAATCAAAAGAATTACTTGGCC
LysSerSerGlnSerLeuLeuTyrSerSerAsnGlnLysAsnTyrLeuAla.
Nucleic acid that synthetic CDR2 has and aminoacid sequence feature are as follows:
TGGGCGTCAACTAGGGAATCT
TrpAlaSerThrArgGluSer.
Aminoacid sequence that synthetic CDR3 has and nucleotide coding sequence feature are as follows:
CAGCAATATTATAGCTATCCGCGGACG
GlnGlnTyrTyrSerTyrProArgThr.
The variable light chain of another synthetic hommization NFS2 has amino acid shown in Fig. 3 and nucleic acid coding sequence feature.This chain has the CDR1 that is same as composition sequence among Fig. 2 and 3 feature.However, prepare the CDR2 that Nucleotide changes by synthetic CDR2 among naturally occurring corresponding CDR2 and Fig. 2.Represent to come from the variation of Fig. 2 composition sequence with beneath stroke of two-wire.Nucleic acid that synthetic CDR2 has and aminoacid sequence feature are as follows:
TGGGCGTCGACTAGGGAATCT
TrpAlaSerThrArgGluSer.
The present invention also comprises Fab fragment or F(ab ')
2Segmental use.A kind of Fab fragment contains the N-terminal part of complete light chain and heavy chain; And F(ab ')
2Fragment is by two fragments that the Fab fragment forms by disulfide-bonded.MAb NFS2 and by its deutero-and describe hereinafter that genetic engineering antibody provides can be by ordinary method, for example by suitable proteolytic enzyme, papoid and/or stomach en-cracking mAb, or obtain Fab fragment and F(ab ' by recombination method)
2Segmental source.This Fab fragment or F(ab ')
2Fragment can obtain from above-described any mAbs, as the protective material of opposing malaria disease substance, particularly falciparum infection in the body.
Can use the variable chains peptide sequence of mouse mAb NFS2, its variable chains peptide sequence and CDRs, function fragment, the fab fragment, with and analogue, obtain the various fusion molecule, particularly genetic engineering antibody of the required fusion rotein of coding with the nucleotide sequence of these molecules of coding, and be used for preparing and using the method for the pharmaceutical composition that contains these molecules.
The nucleotide sequence of the present invention of variable light chain and heavy chain peptide sequence or CDR peptide or its function fragment or its fragment of encoding can the unmodified form be used, or synthetic these sequences are to induce needed modification.Can make from mAb NFS2 or from natural existence or synthetic nucleotide sequence that other required antimalarial protozoon antibody deutero-exsomatize and optionally contain restriction site to promote its insertion or to be connected on the suitable nucleotide sequence in a kind of required antibody skeleton of coding district, be connected with the CDRs of mutagenesis, or merge with the second selected nucleotide sequence that merges the pairing body of encoding.
Consider the degeneracy of genetic code, make up coding variable heavy and light-chain amino acid sequence and CDR sequence of the present invention, the function fragment of the antigen-specific of the total donor antibody of for example Fig. 1-6, and coding and the various encoding sequences of analogue thereof.When with second kind merge the pairing body by operate combine after, can be with of the present invention stripped or synthetic nucleotide sequence or its produced in fragments fusion rotein of coding variable chains peptide sequence or CDRs or its function fragment, promptly chimeric or hommization antibody or other genetic engineering antibodies of the present invention.
Can also insert specific variation with the mutagenesis in the nucleotide sequence in coding CDRs or skeleton district of these sequences, and the nucleotide sequence modified or that merge that will obtain is incorporated in the expression vector.For example, can in the nucleotide sequence of coding CDRs, prepare static nucleotide substitution,, or be similar to the selected skeleton of modification on those nucleotide positions of donor antibody with the insertion of initiative Restriction Enzyme site mutagenesis skeleton.Such sudden change can comprise the sudden change that those insert for antigens with higher binding affinity purpose is provided.
IV. fusion molecule and fusion rotein
Fusion molecule of the present invention can encode engineered antibody, chimeric antibody and hommization antibody.Required fusion molecule can contain and is connected to second by methodology and merges first on the pairing body and merge the pairing body, and this first merges pairing body amino acid sequence coded and have antigen-specific at the plasmodium antibody of repetitive proteins aminoacid sequence and its analogue.Gratifying situation is that first source of merging the pairing body is a kind of selected mAb, for example, and mAb NFS2, the source of Fig. 1 and Fig. 4 amplifying nucleic acid sequence.
A kind of fusion molecule can code pattern 4 in naturally occurring variable heavy chain sequence with and the aminoacid sequence of function fragment or analogue, naturally occurring variable sequence of light chain in can code pattern 1, with and function fragment or analogue, or the aminoacid sequence of one or more NFS2 CDRs.Another kind of typical fusion molecule can be encoded from antibody mAb(as at Fig. 2, describes in 3,5 and 6), the synthetic variable heavy and/or light chain with antigen-specific of plasmodium falciparum antibody.
The feature of gratifying fusion molecule is can encode to contain at least one among the present invention; The preferably weight of all mouse NFS2 antibody and/or the CDRs of variable region of light chain, or the aminoacid sequence of its function fragment or analogue.
Second definition of merging the pairing body as mentioned above, and this second merges pairing body and can comprise coding and the sequence that contains CDR allogenic a peptide, protein or its fragments sequence with antigen-specific of NFS2.The example of such sequence is that the sequence and the selectivity in the significant second antibody of coding district comprises a joint sequence.
If second merges a kind of treatment albumen of pairing body coding itself, or other characteristic antigens, the protein that if this fusion pairing body coding is a kind of to have himself antigen-specific, anti--plasmodium falciparum antigen-specific that the fusion molecule that then obtains can be encoded and second merges the characteristic of pairing body, for example, a kind of functional character, as from recombinant host, secreting, or a kind of treatment feature.
If the second fusion pairing body is to derive from another kind of antibody, for example, any homotype of immunoglobulin (Ig) skeleton or constant region or classification (preferably people) or the like then obtain a kind of engineered antibody.Therefore, for example, a kind of fusion molecule of the present invention can comprise the complete antibody molecule with total length weight and light chain (Fig. 4 and 1).For example, the present invention includes stripped natural existence or synthetic nucleotide sequence, this sequence encoding comes from the variable region sequences of required plasmodium mAbs, the CDR peptide, and its fragment, a kind of any fragment of engineered antibody is as Fab or F(ab ')
2Fragment, a kind of heavy chain homodimer, or as any basic recombinant fragment of Fv or single-chain antibody (SCA), or coding has and is same as selected mAb, for example, specific a kind of proteinic any other sequence of plasmodium mAb NFS2.
First merges the pairing body can also unite use with the effector agent of above definition, adopts ordinary method to be connected on this effector agent by merging the pairing body with first after operating.For example, be adsorbed onto on the coding NFS2 nucleic acid by covalency bridged bond structure.
Adopt any appropriate method, for example, adopt conventional covalency or ionic linkage, protein fusion, or allos bi-functional cross-linking agent, for example, and carbodiimide, glutaraldehyde or the like can merge the pairing body with first and merge or bonding with the second selected fusion pairing body.Combine with a kind of effector agent if first merges the pairing body, can adopt nonprotein, habitual chemical linking agent is with the fusion of anti-plasmodium falciparum aminoacid sequence or be connected on the effector agent.These technology are known in this area, and have carried out simple description in the chemistry of routine and biological chemistry textbook.
In addition, can also provide the conventional ellipse property joint sequence of desired number spatial to be building up in the fusion molecule between pairing body and the effector agent with merging at this fusion pairing body or first easily.The method that designs this joint also is that those skilled in that art know.
Express these fusion molecule and obtain fusion rotein of the present invention.A kind of fusion rotein of specific required type comprises engineered antibody, in this antibody, with minimum degree, use from one or more donor monoclonal antibodies and can lighten and/or the similar portions of heavy chain substitutes the variable heavy and/or light chain district fragment of a kind of acceptor mAb, this donor monoclonal antibody comprises the plasmodium mAbs that describes herein, as NFS2.
Specific required engineered antibody is a kind of hommization antibody, and the CDRs that comes from required donor mouse mAb therein is inserted into a kind of skeleton district of people's antibody.A kind of preferred donor antibody is a kind of antibody at the plasmodium antigens determinant, preferably is specific to the antibody of the iteron antigenic determinant of plasmodium falciparum, preferably is specific to the antibody of the iteron antigenic determinant of plasmodium falciparum.A kind of specific preferred antibody that supplies has the whole of NFS2 or part variable region amino acid sequence.In these hommization antibody, will come from plasmodium antibody heavy chain and/or variable region of light chain one, two or preferably three CDRs be inserted into the skeleton district of selected people's antibody, to substitute the natural CDRs of this back one antibody.
Preferably, changed in the heavy and light intrachain variable region of people by CDR is alternative.Therefore, the engineered antibody of this hommization preferably has natural human antibody or its segmental structure.Such hommization antibody can comprise or not comprise that minimum change in the light and/or heavy variable domain skeleton of the acceptor mAb district is with reservation donor mAb binding specificity.This hommization antibody has for resisting and treat the required overall characteristic of infectious plasmodium falciparum malaria among the human or animal effectively.
Remaining engineered antibody can obtain from any suitable acceptor human normal immunoglobulin.A kind of suitable people's antibody can be a kind of from the routine data storehouse, for example, and Kabat database, Los Alamos database, and the antibody of that select and Nucleotide donor antibody and amino acid sequence homologous in the Swiss Protein Data Bank.Having people's antibody with skeleton district (based on a seed amino acid) homonome of donor antibody is well-suited for and inserts donor CDRs and heavy chain constant region and/or weight chain variable skeleton district are provided.Choose the suitable receptor antibody that to supply the constant or variable skeleton of light chain district in the same way.Should be noted that and do not require that receptor antibody weighs and light chain must derive from same person antibody.
Gratifying situation is, from human normal immunoglobulin classification and homotype, as IgG(hypotype 1-4), IgM, IgA and IgE select allos skeleton and constant region.But, receptor antibody does not need to include only the human normal immunoglobulin sequence, for example can also make up a kind of group, wherein the dna sequence dna of encoding part human normal immunoglobulin chain is fused on a kind of DNA of aminoacid sequence of coding one peptide species effector agent or acceptor molecule.
As an example, substitute the part nucleotide sequence of the variable region of light chain of a kind of acceptor mAb that encodes at least by synthetic nucleic acid sequence with the CDRs in coding NFS2 variable light chain district or its function fragment, and substitute a kind of acceptor mAb that encodes at least with the CDRs in variable heavy chain district of coding NFS2 or the nucleotide sequence of its function fragment, as a kind of part nucleotide sequence of variable region of heavy chain of people's antibody, and a kind of engineered antibody of encoding.The hommization antibody that obtains has the antigen-binding specificity feature of mAb NFS2.
Perhaps change a kind of method, engineered antibody of the present invention (or other monoclonal antibodies) can be adsorbed on its a kind of effector agent or acceptor molecule.Perhaps, can produce a kind of engineered antibody of the present invention, wherein substitute a kind of Fc fragment or CH3 district of complete antibody molecule with a kind of enzyme or lps molecule with the step of recombinant DNA technology.
Those of skill in the art understand in this area, by changing variable region amino acid, but not necessarily must influence the specificity of donor antibody and further modify above-mentioned engineered antibody.Can consider to be used in variable region skeleton or CDRs or two kinds of heavy and light chain amino acids of other amino acid replacements that structure is interior.It is effective effectively using such engineered antibody during malaria infection (for example, by plasmodium falciparum) in the opposing human body.
In addition, the fusion rotein of the chimeric antibody of definition in the invention provides as mentioned.These antibody comprise for example Fig. 1 and 4 of skeleton district because the heavy chain and the variable region of light chain of whole donor antibody can be provided, and with the constant region of corresponding two chains of being used to be fused to receptor antibody, thereby are different from above-described hommization antibody.
V. the preparation of protein and antibody
Use gene engineering, can make up fusion molecule of the present invention, recombinant antibodies or fusion rotein satisfactorily by recombinant DNA method.Also can prepare other concrete materials of the present invention, for example, make up the chimeric or hommization antibody of above mentioning, the light and heavy chain of synthetic, the nucleotide sequence of CDRs and these materials of encoding with same or similar techniques.
Use CDRs and one or more the selected people's antibody of the present invention specific concrete composition light and that heavy chain skeleton district prepares of mouse NFS2 to list among the embodiment 3.Be described below simply, use the ordinary method clone to produce the hybridoma of mouse NFS2 antibody, and obtain its heavy and cDNA variable region of light chain with the known technology of those skilled in that art, for example, at Sambrook et al., Molecular Cloning(A Laboratory Manual), the 2nd edition, Cold Spring Harbor Laboratory(1889) the middle technology of describing.Utilize the PCR primer to obtain the variable region of NFS2, and use known Computer Database, for example, Kabat differentiates in the storehouse this CDRs, and compares with other antibody.
Use same database, for example homology skeleton district of the variable region of heavy chain of Kabat storehouse discriminating people antibody, and selection and NFS2 have people's antibody of homology as receptor antibody.Design contains the synthetic weight chain variabl area sequence of NFS2 CDRs in people's antibody skeleton district, nucleotide substitution can take place or not take place to be used as restriction site in the skeleton district of this sequence.Synthesize so sequence of design by overlapping polynucleotide, by polymerase chain reaction (PCR) increase this sequence and error recovery.
With suitable light chain variable skeleton district of the same manner design, produce two synthetic light chain variable sequences that contain NFS2CDRs.Referring to, Fig. 2 and 3.As noted above, the source of light chain is not a restriction factor of the present invention.
Method below adopting is making up fusion rotein of the present invention and engineered antibody, has preferably used these synthetic to lighten and/or the CDRs of sequence of heavy chain and mAb NFS2 and their nucleic acid coding sequence during people's antibody.Pass through ordinary method, obtain the dna sequence dna in the heavy or light chain district of coding donor antibody variable, wherein need the CDRs in the light and/or heavy chain variable domain skeleton district of acceptor mAb and those essential parts at least to keep the donor mAb binding specificity and and need derive from the residual immunity sphaeroprotein derivative moiety of the antibody chain of human normal immunoglobulin.
By after the operation with the encoding sequence of these fusion roteins with can control it and in host cell, duplicate and express also/or from the host adjusting control sequence of excretory routine combine and prepare the expression vector or the recombinant plasmid of routine.Arbitrary technician can easily choose such adjusting sequence in this area, and the invention is not restricted to such adjusting sequence.Regulate sequence and comprise promoter sequence, CMV promotor for example, and with arbitrary technology in this area from the signal sequence of Antibody Preparation.
First carrier can contain the nucleotide sequence of the polypeptide of the derived light chain of encoding.Equally, prepare second expression vector, this carrier has the same dna sequence dna of the light or heavy chain of complementary antibody of coding.Preferred situation is, in these carriers, at least the CDRs(of variable region and for keep acceptor mAb that the donor mAb binding specificity needs gently and/or those essential parts of heavy chain variable domain structural area) derive from donor antibody and the remaining antibody chain that comes from immunoglobulin (Ig) partly obtains from human normal immunoglobulin.Preferred situation is that these second expression vectors are same as first carrier except that encoding sequence is different with the selected marker thing, functionally express to guarantee each bar peptide chain.
In the selectable method of another kind be, also can be only with single carrier of the present invention, this carrier contains the sequence of coding light chain and heavy chain polypeptides derived.The DNA of encoded light and heavy chain comprises that also cDNA or genomic dna or both have in this encoding sequence.
Use first and second carriers (or single carrier) and the conventional selected host cell of technology cotransfection, contained the host cell of the transfection of the present invention of reorganization or gently synthetic and heavy chain with preparation.Cultivate transfectional cell to prepare engineered antibody of the present invention with routine techniques then.Adopt suitable detection method, as the ELISA detection method, and the sporozoite of describing among following embodiment invasion suppresses (ISI) detection method can be screened the composition that contains reorganization heavy chain and/or light chain from culture hommization antibody.Available similar routine techniques makes up other fusion roteins of the present invention.
Therefore, the present invention also comprises the recombinant plasmid of the encoding sequence that contains fusion molecule of the present invention or engineered antibody.Adopt a kind of like this carrier of routine techniques preparation, and suitably contain above-described dna sequence dna and by a kind of suitable promotor on the dna sequence dna that is connected to the coding engineered antibody after the operation.For help routine techniques with above-mentioned carrier transfection in mammalian cell.
By arbitrary those skilled in the art can be chosen in the inventive method and composition in making up the clone and the subclone step in the suitable carrier that uses.For example, the pUC cloning vector sequence of available routine.Employed a kind of carrier is pUC19, and this carrier can be by commercial sources from supply company, as Amershan(Buckinghamshire, United Kingdom) or Pharmacia(Uppsala, Sweden) locate to buy.In addition, any can duplicating easily has abundant cloning site and marker gene, and the carrier of operation processing easily can be used for the clone.Therefore the selection to cloning vector is not a restrictive factor of the present invention.
Equally, can from any conventional carrier, select the carrier that is used for the present invention's expression engineered antibody by arbitrary technician in this area.This carrier also contains selected adjusting sequence, and this sequence combines with the dna encoding series of operations of immunoglobulin domain and the directing heterologous dna sequence dna of energy duplicates in selected host cell and express this adjusting sequence such as CMV promotor.These carriers contain the dna sequence dna of above-described coding engineered antibody or other fusion roteins.Method for changing can be incorporated into carrier in the selected immunoglobulin sequences of modifying for the ease of the operation required restriction site of insertion.
The feature of this expression vector also is to have and is applicable to the marker gene that increases the allogeneic dna sequence expression, for example, and Mammals dihydrofolate reductase gene (DHFR) or neomycin resistance gene (neo
R).Other preferred carrier sequences comprise as a poly a-signal sequence from Trobest (BGH), and the betaglobulin promoter sequence (β-glupro).Adopt the technology that those of skill in the art know in this area can synthesize the expression vector that uses among the present invention.
All can obtain the various compositions of above-mentioned carrier from natural source or with the material that the known technology synthetic is used for instructing recombinant DNA to express selected host, for example replicon is selected gene, adds hadron, promotor or the like.For above-mentioned purpose can also be selected the Mammals that is used for known in the art, bacterium, insect, the suitable expression vector of other of many types of yeast and expressed in fungi.
It is that Mammals carrier Pfhzhc2-3-Pcd and Pfhzlc1-1-Pcn(see Fig. 7 and 8 that being used to of using in the following example expressed heavy and two typical expression vectors sequence of light chain of synthetic).But, the invention is not restricted to the carrier that uses these to give an example based on pUC19.
The present invention also comprises the recombinant plasmid cells transfected system with the encoding sequence that contains the engineered antibody described by the present invention or other fusion roteins.Because with the cloning of these cloning vectors and the host cell of other operations also is conventional the use.But the most satisfied is, will come from other steps that the cell of colibacillary various bacterial strains is used for duplicating of cloning vector and makes up at mAbs of the present invention.
Be used to express preferably eukaryotic cell of engineered antibody of the present invention or other proteinic appropriate host cell or clone, and mammalian cell most preferably, as Chinese hamster ovary celI or marrow like cell, can be with other primates zooblasts as host cell, and the optimal people's cell that is to use, glycosylation pattern is modified thereby this protein can be chosen.In addition, can use other eukaryotic cells.The selection of suitable mammalian host cell, and be used for transforming, cultivate, amplification, the screening and the preparation of product and the method for purifying all are known in the art.Referring to, for example, Sambrook et al., as mentioned above.
Bacterial cell proves that also it is to be used as the proper host cell of expressing reorganization mAbs of the present invention.But trending towards owing to expressed protein in bacterial cell is folding or non-correct folded form or non-glycosylated form, thereby must filter out the reorganization mAb of the bacterial cell generation that has kept antigen binding capacity.If by the bacterial cell expressed protein, then this bacterial cell is satisfied host with correct folded form production.For example, the various coli strains that are used to express are host cells of knowing in the biological technical field.Bacillus subtilus, streptomycete, the various bacterial strains of other bacillus etc. also can use in the method.
If desired, the known yeast strain cell of those of skill in the art in this area and insect cell and virus expression systems can also be used as host cell.Referring to, as Miller et al., Genetic Engineering, 8:277-298, Plenum Press(1986) and be incorporated herein for referencial use.
Can be used for making up the universal method of carrier of the present invention, produce the required transfection method of host cell of the present invention, and prepare fusion rotein of the present invention from above-mentioned host cell, preferably the necessary cultural method of engineered antibody all is a common technology.Equally, in case after preparing, can comprise ammonium sulfate precipitation method according to the standard method in this area, the affinity column column chromatography, gel electrophoresis or the like is purified fusion protein from cell culture, engineered antibody preferably of the present invention.These technology are that those skilled in that art are known, and do not limit the present invention.
Adopt the detection method that is applicable to selected cause of disease to check the external activity of engineered antibody then.Use present conventional ELISA detection scheme can assess the situation that combines of engineered antibody and R32tet32 antigenic determinant qualitative and quantitatively.Can also be used in the ISI detection method that embodiment 6 describes.Can finish similar a kind of detection method-inhibition invasion hepatocellular detection method (ILSDA) [S.Mellouk et al., Bull.WHO, Suppl.68:52-58(1990)].In addition, before people's clinical study of carrying out subsequently, exist removing mechanism engineered antibody still can remain in the body lastingly with estimation although can also be used in the detection method verification efficiency of setting up at present in the SCID mouse model.
The following examples have illustrated the method that is used to make up from mouse mAb NFS2 deutero-hommization antibody.According to the method for description from this Antibody Preparation hommization antibody, the arbitrary technician in this area also can be from the antibody of other malaria of describing herein, and variable region sequences and CDR peptide make up hommization antibody.Be identified as the variable region skeleton preparation engineering antibody of " self " with the antibody receptor that might have been changed.In the skeleton of variable region, carry out small modification and can cause the huge increase of antigen bonding force, and estimable increase does not take place in the immunogenicity of acceptor.Above-mentioned engineered antibody can protect human body to avoid falciparum infection effectively passively.
VI. treatment/preventive use
Can be with the fusion rotein of describing herein, especially above the engineered antibody of Miao Shuing, function fragment, analogue and other protein and peptide are used as preventive, it can offer the passive immunity to pathogenic infection of experimenter's short-term, from this pathogenic agent, for example, plasmodium falciparum derives the original antigen material.By immunoglobulin (Ig) is combined with pathogenic agent, and remove by the scavenger cell of normal function subsequently that this binding mixture can produce because the provide protection of using engineered antibody of the present invention to provide.Therefore, engineered antibody of the present invention, when making when being suitable for preventing preparation that purpose uses and prescription, to carrying out the people of short term contact with this pathogenic agent, for example, and might be the traveller of region of disease travelling, visitor or soldier are in demand.
Therefore; the invention still further relates to prophylactic treatment needs the method for falciparum infection in the human body of this treatment, this method to comprise to the human body that might contact with the plasmodium kind to use one or more engineered antibodies that comprise description herein or the antibody of other fusion roteins or their segmental a kind of effective protection dosage.
Can also engineered antibody of the present invention or its segmental fusion rotein and other antibody will be comprised, especially can unite use with the people mAbs that other antigenic determinants that cause this disease react, the directly anti-antigenic determinant that causes disease of engineered antibody wherein of the present invention.Equally, can also use the mAbs that reacts with other antigenic determinants that can cause disease in selected animal with the animal medicinal composition form, antibody wherein of the present invention is directly at the antigenic determinant that causes disease.Any not inconsistent but active antibody with plasmodium antibody of the present invention, for example the antibody at other malaria periods or different antigenic determinants can both be used for these compositions.
It is believed that preventive of the present invention can provide provide protection satisfactorily in the time that contacts about 4 days-8 weeks with pathogenic agent, and do not need to strengthen using the dosage of this reagent." short-term " definition is meant the relative extended period of recombinant antibodies of the present invention in people's body-internal-circulation.
Can to be any energy discharge suitable pathways to the host with this medicament to the medication of preventive of the present invention.Comprise engineered antibody, and segmental fusion rotein, and medicinal compositions of the present invention is specially adapted to parenterai administration, also promptly subcutaneous, muscle or intravenously.But this pharmacy optimization adopts the intramuscular injection administration.
TGGGCGTCGA CTAGGGAATC T 21
(2) data of SEQ ID NO:41:
(ⅰ) sequence signature:
(A) length: 7 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:41
Trp Ala Ser Thr Arg Glu Ser
1 5
(2) data of SEQ ID NO:42:
(ⅰ) sequence signature:
(A) length: 366 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..366
(ⅹ ⅰ) sequence description: SEQ ID NO:42
Pass through parenterai administration to the of the present invention a kind of protein of about 20mg/Kg or antibody, preferably muscle (i.m.) and possible be vein (i.v.) administration.After the contact pathogenic agent, can repeat this dosage with suitable interval.
Can be with antibody described herein, engineered antibody or its fragment freeze-drying are stored, and are formulated in the suitable carriers again before use.Verified these technology are effectively to the immunoglobulin (Ig) of routine, and can be with freeze-drying known in the art and compound method again.
Dosage level that use treatment doctor selectes and pattern are with the administration of aforementioned pharmaceutical compositions single or multiple.In any case, the amount that pharmaceutical composition of the present invention should provide enough opposings effectively to infect required engineered antibody of the present invention.
The following example is described the structure of typical engineered antibody, and the expression in suitable carrier and host, and does not constitute limitation of the scope of the invention.Except that other explanations are arranged, define all amino acid with conventional code or full name.Removing has other explanations, all restriction enzymes, and plasmid and other reagent and material obtain by commercial sources.The clone that all are general connects and other recombinant DNA technologies are according to Sambrook et al., described in the past, or its first version is described and finished.
By with the duplicate injection of plasmodium falciparum sporozoite in mouse, B cell and myeloma cell line are merged can prepare mouse IgG mAb NFS2 subsequently.The feature of mouse mAb NFS2 is the antigen-binding specificity that has at the proteinic iteron of plasmodium falciparum CS.Distinguishingly be that NFS2 mAb is attached on the antigenic determinant Pro Asn Ala Asn Pro Asn.This antibody can also be attached to of iteron big or the eclipsed antigenic determinant on also be possible.
In the vitro detection method, this mouse mAb stops sporozoite to invade human liver cell and liver cancer cell.In mouse model similarly antibody the passive protection of antibody malaria is provided and have been found that this antibody be highly effective [referring to; R.A.Wirtz et al. for example; Bull WHO; 65:39-45(1987); be incorporated herein for referencial use], this antibody can obtain from U.S. naval medicine research institute.
Cloning and the order-checking of embodiment 2 NFS2
Use Favaloro et al., method Meth.Enzymal., 65:718-749(1980) prepare kytoplasm RNA from NFS2 and hybridoma cell line.Respectively at the heavy (V of Ig
H) and light (V
L) use following primer in chain variable region cDNAs synthetic.This V
LPrimer, #2580 and #2789 extend direct puncture from the Hind II and cross the Xba I, and make this primer arrive the conserved regions of mouse RNA.
HindIII
#2580:5'CCAGATGTAAGCTTCAGCTGACCCAGTCTCCA3'
PvuII
XbaI NaeI
#2789:
5'CATCTAGATGGCGCCGCCACAGTACGTTTGATCTCCAGCTTGGTCCC3'
V
HPrimer, #2621 and #2853 extend up to the Pst I from the Kpn I, and make this primer arrive the conserved regions of mouse RNA.
KpnI XhoI
#2621:5'GGGGTACCAGGTCCA(A/G)CT(G/T)CTCGAGTC(A/T)GG3'
PstI
#2853:5'GCCTGCAGCTAGCTGAGGAGACGGTGACCGTGGTCCCTTGG-
NheI
CCCCAG3'
According to Saiki et al., Science 239:487-491(1988) describes, and finishes PCR on the RNA template.For PCR, according to the employed primer of discriminating mentioned above.For V
HPcr amplification, DNA/ primer miscellany is made up of this primer of 5 μ lRNA and 0.5 μ M.For V
LPcr amplification, the DNA/ primer mixture is made up of this primer of 5 μ l RNA and 0.5 μ M.The various dATP that in these mixtures, add 250 μ M, dCTP, dGTP and dTTP, 10mM Tris-HCl pH8.3,50mM KCl, 1.5mM MgCl
2, 0.01%(W/V) gelatin, 0.01%(V/V) polysorbas20,0.01%(V/V) the Ampli Taq[Cetus of Nonidet P40 and 5 units].Sample is carried out 25 PCR thermal cyclings, and each circulates in 94 ℃, 30 seconds; At 55 ℃, 30 seconds; 72 ℃, 45 seconds; And carried out 5 minutes and finish at 72 ℃.In order to clone and to check order, on the low melting-point agarose gel and the V that adopts the amplification of Elutip-d column chromatography [Schleicher and Schuell-Dussel, Germany] purifying
HDNA, and be cloned into pUC18[New England Biolabs].People such as Maniatis, with above having described clone and connection side's science of law commonly used.
With V
HDNA is cloned among the pUC18 of same digestion with KpnI-Pst I pieces.With V
LDNA is cloned among the pUC18 with same enzymic digestion with Hind III-Xba I pieces.The employing dideoxy method [Sanger et al., Proc.Natl.Acad.Sci.USA, 74:5463-5467(1977)] and use T7DNA polysaccharase [US Biologicals] to measure representative sequence of cloning.From NFS2V
HAnd V
LThe sequence in district, according to people such as Kabat, at " Sequerices of Proteins of Immunological Interest ", US Dept of Health and Human Services, US Government Printing office(1987) method described in uses a computer auxiliary and other V
HAnd V
LThe CDR sequence is explained in the arrangement contrast of sequence.The weight of NFS2 and the CDRs of light chain list in above and differentiate in Fig. 1.
The following examples have been described a kind of typical engineered antibody preparation.Use is by other plasmodium antibodies or other antipathogen antibody of ordinary method preparation, and making uses the same method is used for preparation engineering antibody.
The donor CDRs source that is used for preparing these engineered antibodies is the foregoing description 1 and the 2 mouse mAb that describe, NFS2.Use through the variable skeleton of the NFS2 district of order-checking and retrieve the homology skeleton district of Kabat database once more with identification people antibody.There is 80% homology in the skeleton district [H.Dersimonian et al., J.Immunol., 139:2496-2501(1987)] that measures from people SLE patient's B cell myeloma cell line 18/17 antibody that obtains approximately with NFS2 variable heavy chain skeleton district.
Possessing mouse NFS2 CDRs(embodiment 2) and the situation of people's antibody 18/17 sequence under, preparation synthetic variable region of heavy chain, the performing PCR of going forward side by side is to fill up and DNA amplification.Adopt synthetic NFS2 CDR sequence of following overlapping oligonucleotide and 18/17V
HThe skeleton district:
TAGTGAAGAATTCGAGGACGCCAGCAACATGGTGTTGCAGACCCAGGTCTTCATTTC
TCTGTTGCTCTGGATCTCTGGTGCCTACGGGGAGGTGCAG(Base 1-97);
GCTAGCGGATTCACCTTTAGCAGCCATGTCGGACCCCCCAGGGACTCTGAGAGGACA
CGTCGATCGCCTAAGTGGAAATCCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGG
AAAGGTCTAGAGTGGGTCTCAGAAATCTTTATAGTGATGGTGGTAGTTAC(Base
158-259);
GAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTCTGTTAAG
GTTCTTGTGCGACATAGACGTTTACTGCAGTATATTACTGTGCGAAACTCATCTACT
ATGGTTACGACGGGTATGCTATGGACTAGCTGCCCATACGATACCTGATC(Base
316-421);
TTCTTGAAAGCTTGGGCCCTTGGTACTAGCTGAGCTCACGGTGACCAGGGTACCCTG
GCCCCAGTAGTCCATAGCATACCCGTCG(Base 484-400);
CATTTGCAGATACAGCGTGTTCTTGGAATTGTCTCTGGATATCGTGAACCGGCCCGT
CACAGTGTCTGGATAGTAGGTGTAACTACCACCATCACTAATTTC(Base 337-
236);
CTAAAGGTGAATCCGCTAGCTGCACAGGAGAGTCTCAGGGACCCCCCAGGCTGTACC
AAGCCTCCCCCAGACTCGAGCAGCTGCACCTCCCCGTAGGCACC(Base 177-
77).
These primer annealings to also filling up DNA with the Taq polysaccharase together, are carried out pcr amplification with following 5 ' primer CCGCGAATTCGAGGACGCCAGCAAC and 3 ' CCGCAAGCTTGGGCCCTTGGTACTAGCT.. subsequently.
Correction is inserted into any mistake in the sequence that is detected by PCR.In addition, the nucleotide substitution of inserting conservative property in the skeleton district is applicable to the selected restriction site of enzymatic cutting with introducing.By at Fig. 2, the frame that adds in 3,5 and 6 on the sequence is partly represented these variations in the skeleton district.In addition, most mouse and people's antibody had alkaline residue before CDR3.Because the variable heavy chain of NFS2 has a non-alkaline residue Ser before CDR3, the alkaline residue (Lys) before the CDR3 of disappearance acceptor is also alternative with preparation heavy chain Pfhzhc2-3 with Ser.
Obtain two synthetic variable region of heavy chain, called after Pfhzhc2-3 and Pfhzhc2-4.In Fig. 5 and 6, describe these sequences in detail.Each of these synthetic variable region of heavy chain has one or more Nucleotide, or amino acid feature inequality.For example, Pfhzhc2-3 has a Ser at 98; And Pfhzhc2-4 has a Lys on 98.In addition, these variable region of heavy chain are identical.
For suitable light chain variable skeleton district, adopt same quadrat method, use is at H.G.Klobeck et al., and Nucl.Acids Res. knows the NFS2 light chain CDRs of others antibody and light chain variable frame sequence in 13:6515-6529(1985) to prepare suitable synthetic sequence of light chain.The oligonucleotide that uses is as follows:
TAAGCGGAATTCGTAGTCGGATATCGTGATGACCCAGTCTCCAGACTCGCTAGCTGT
GTCTCTGGGCGAGAGGGC(Base 1-75);
TTACTTGGCCTGGTATCAGCAGAAACCCGGGCAGTCTCCTAAGTTGCTCATAGTTTT
CTTAATGAACCGGACTTACTGGGCGTCAACTAG(Base 130-198);
GACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTATACTA
CTGCTGTCTAAAGTGTCAGCAATATTATAGCTATCC(Base 241-321);
CAGTTGGAAAGCTTGGCGCCGCCACAGTACGTTTGATCTCCACCTTGGTCCCTCCGC
CGAACGTCCGCGGATAGCTATAATATTGC(Base 389-304);
GTGAAATCTGTCCCAGACCCGCTGCCACTGAATCGGTCAGGTACCCCAGATTCCCTA
GTTGACGCC(Base 252-187);
CAGGCCAAGTAATTCTTTTGATTGCTCGAGTATAAAAGGCTCTGAGAGCTCTTGCAG
TTGATGGTGGCCCTCTCGCCC(Base 141-64).
Describe as mentioned, primer annealing is arrived together, and fill up this DNA, use following primer subsequently with the Taq polysaccharase
5'GCGGAATTCGTAGTCGGATATCGTGATGAC and
The 3'TGGAAAGCTTGGCGCCGCCACAGTACGTTTGATC primer
Carry out pcr amplification.
Contain two synthetic light chain variable sequences of NFS2CDRs and be referred to as Pfhzlc1-1 and Pfhzlc1-2 according to the method design of above describing that is used for synthetic heavy chain and synthetic two.This two sequences only has an amino acid inequality on 49 of aminoacid sequence.Pfhzlc1-1 has a Ser on 49; Pfhzlc1-2 has a Pro on same position.
In a kind of building process of the hommization antibody that exemplifies, used these synthetic can lighten and/or sequence of heavy chain.We expect that any synthetic heavy chain can successfully combine with any synthetic light chain to prepare a kind of useful hommization antibody.
In order to prepare hommization antibody, Pfhzhc2-3(sees Fig. 6 for the weight chain variable sequence), synthetic following signal sequence on this variable region:
ATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTAC,
This sequence encoding:
MetValLeuGlnThrGlnValPheIleSerLeuLeuLeuTrpIleSerGlyAlaTyr.
For synthetic light chain variable sequence Pfhzlcl-1(Fig. 2), digest this construct with EcoR I and EcoR V, and identical signal sequence is connected on the variable sequence.Other signal sequences of those skilled in that art are also known, and be can be used for substituting this type sequence.
Synthetic and confirm to select for use the human IgG of selecting of making heavy and light chain by PCR
1The constant region of antibody.Then these constant region sequences are inserted in the expression vector based on pUC19.Adopt ordinary method [Maniatis et al. afterwards, together above] will contain the signal of light and heavy chain and the above-mentioned synthetic variable construct of variable region and be inserted in these expression vectors that contain CMV promotor and constant region based on pUC19, and the heavy and light chain constant region fusion with the people who inserts previously in framework.Therefore, after being inserted into the synthetic variable region in these expression vectors, be created in the plasmid that shows in Fig. 7 and 8.Then with these plasmid co-transfections in selected host, and after cultivation, adopt the ELISA method of describing in the following example 4 to analyze the antibody activity of this substratum.
Use same technology, and synthetic sequence of heavy chain Pfhzhc2-3(Fig. 6) and synthetic sequence of light chain Pfhzlcl-2 make up another kind of representational hommization antibody.
Amino acid whose variation among the variable region of the skeleton of the original murine antibody NSF2 that describes among the mensuration embodiment 1 and 2 and the Pfhzhc2.3, and prepare several variations to improve the preservation level of original antibody configuration.
On 49 amino acids sites, the Ser of hommization heavy chain Pfhzhc2.3 is changed into Ala, Ala is the amino acid that is present on the corresponding position of this natural mouse NSF2.This alternative Process has been used conventional gene engineering, and for example, preparation contains the synthetic DNA fragment of suitable Nucleotide variation to change amino acid.With the fragment of Xba I and EcoR V digestion Pfhzhc2.3, and will carry the synthetic fragment that the Nucleotide of coding Ala rather than Ser codon changes and insert so that amino acid replacement to take place.The synthetic heavy chain that obtains is called Pfhzhc2.6.
Express synthetic this heavy chain according to the previously described method that is used for the synthetic heavy chain of Pfhzhc2.3.The expression plasmid of this hommization sequence of heavy chain is equal to the expression plasmid of describing among Fig. 7 basically, and different is to have any different just like preceding described single amino acids.
Equally, the hommization antibody of forming by Pfhzhc2.6 heavy chain and Pfhzlc.1 light chain and Pfhzhc2.6 heavy chain and Pfhzlc1.2 light chain by means of cotransfection mammalian cell preparation, and with the ELISA detection antibody activity of description among the embodiment 5 hereinafter.
The same quadrat method of utilizing design to be used to obtain minimum variable region backbone modification can prepare the antibody that is specific to plasmodium falciparum of other high-affinities.This method comprises the change and the test procedure of following order:
(1) except that 49 amino acids change, known other single skeleton amino-acid residues that play key effect in interacting with CDRs that will be present in original antibody and the generation CDR alternate engineered antibody compare.For example, will be present in heavy chain amino-acid residue in original (donor) and the engineered antibody (Kabat numbering; Referring to Kabat et al., together above) compare.It is believed that a residue on this site interacts by means of the constant heavy chain CDR residue on a salt bridge and 94.
If in the skeleton of donor antibody and in the skeleton at engineered antibody, then preparation does not contain the another kind of heavy chain gene of this project antibody for amino acid.Under reverse situation, do not have in donor antibody if on a position of engineered antibody skeleton, contain a residue, then prepare a kind of original amino acid whose another kind of heavy chain gene that on that site, contains again.Before further analyzing, detect the engineered antibody of the another kind of plasmid of preparation based on this with the preparation high-affinity.
(2) will be present in original antibody and take place in the CDR alternate engineered antibody according to Kabat(referring to people such as Kabat, the same) four intra-residue skeleton amino acid of the CDRs of definition compare.If there is difference, then for each zone, the specific amino acids in that zone is substituted the amino acid of the respective regions in the engineered antibody, so that engineering group in a small amount to be provided.The other plasmid that detects generation on this basis then is with preparation high affinity antibody.
(3) framework residue original and that take place in the CDR alternate engineered antibody is compared, and indicate at electric charge, there is huge constant residue size or hydrophobicity aspect.With the amino acid preparation substitute plasmid of the single sign of the corresponding amino acid represent of original antibody, test this substitute plasmid based on this with the generation high-affinity antibody.
By plasmid DNA s transient transfection being detected the expression of synthetic heavy chain and sequence of light chain in the monkey COS cell.That report below is the result of Pfhzhc2-3 and Pfhzlc1-1.The plasmid of 10 micrograms is mixed, and use ethanol sedimentation.This DNAs is dissolved in the Tris buffer saline (TBS) and mixed, mixture is added to 3-4 * 10 that grow in the T25 flask with DEAE-dextran (400 μ g/ml final concentration)/chloroquine (0.1mm)
5In the COS cell, and 37 ℃ of cultivations 4 hours.Make cell shock 1-2 minute and after with the PBS washing, cell is cultivated in the serum-free growth medium with being dissolved in 10%DMSO in the phosphate-buffered saline (PBS).72 hours (3 days samples) are collected substratum and are added fresh culture after the transfection, and 120 hours (being called 5 days samples) collected culture after transfection.
For more various antibody are chimeric and binding affinity hommization antibody, by finishing large-scale COS transfection as mentioned above.For each antibody, use 200 μ g heavy chain plasmids and 200 μ g light chain plasmid DNA s with transfection 2.5 * 10
7The COS cell of sum.Store the substratum of collecting (3 days and 5 days), catch ELISA with Fc and detect antibody expression.With Amicon substratum is concentrated into 6ml.The amount of antibody changes in 9mg/ml to 25mg/ml scope in the substratum of storing.Utilize the binding affinity of ISI and the more above-mentioned concentrated sample of ILSDA detection method.
Adopt conventional elisa technique to detect the hommization antibody that in the substratum of the plate well that contains the transfection clone, exists.Goat Anti-Human IgG(Fc specificity with every hole 0.1 μ g degree of depth) antibody [Sigma, St.Louis, MO] wraps by the droplet plate, spends the night at 4 ℃.Using PBS(pH7.5) after the washing, in the future 50 μ l substratum in the hole of self-contained transfectant are added in each droplet hole, keep 2 hours under the room temperature.Vacate each hole then, with PBS washing and to every hole add 1/1000 dilution of 50 μ l peroxidase-conjugated goat Anti-Human IgG antibody (BioRad, Richmond, CA).Then flat board was at room temperature cultivated 1 hour.Vacate each hole then, wash with PBS.In every hole, add 2.2 of 100 μ l '-azino-two (3-ethyl-phenyl thiazole sulfonate moiety salt (6)].Reaction was at room temperature continued 1 hour.Adopt spectrophotometer to detect the absorption value at 405nm place then.Detect the ability of hommization antibody conjugates to the plasmodium falciparum circumsporozoite protein in the substratum in the hole of containing the transfection clone by ELISA.Wrap by titer plate with the R32tet32 that intestinal bacteria produce with every hole 0.1 μ g concentration, spend the night at 4 ℃.After the PBS washing, 50 μ l substratum in the hole of self-contained transfectant are added in each microtitre hole in the future, keep 2 hours under the room temperature.Vacate each hole then, with PBS washing and to every hole add 1/1000 dilution of 50 μ g peroxidase-conjugated goat Anti-Human IgG antibody (BioRad, Richmond, CA].Then flat board was at room temperature cultivated 1 hour.Vacate each hole then, wash with PBS.In every hole, add 2.2 of 100 μ l '-azino-two (3-ethyl-phenyl thiazole sulfonate moiety salt (6)].Reaction was at room temperature continued 1 hour.Adopt spectrophotometer to detect the absorption value at 405nm place then.
In preliminary study, compare with Pfhzhc2-3 heavy chain construct, the affinity of observing the hommization antibody among the embodiment 4 that contains Pfhzhc2-6 heavy chain construct improves.
The structure of embodiment 6 chimeric antibodies
Basically make up chimeric antibody of the present invention by above describing.A kind of chimeric antibody contains to be selected as heavy chain [H.Dersimonian et al., J.Immunol., 139:2496-2501(1987) and light chain [Klobeck et al., Nucl.Acids.Res., variable skeleton of natural mouse NSF2 13:6515-6529(1985)] and the CDR zone on people's constant region, the situation of exception are that the RNA of the murine antibody that will obtain from the NFS2 hybridoma carries out PCR and obtains the variable region and by the synthetic human IgG of overlapping oligonucleotide
1The whole constant region of antibody also increases by PCR.Proofread and correct any mistake of inserting owing to PCR.The method that is used for hommization antibody by foregoing description is expressed chimeric heavy chain and the chimeric light chain that obtains.
Because this chimeric antibody has the active feature that is equal to natural murine antibody basically, thereby is favourable, but this antibody contains the enough human sequences that can be used for treating human body diseases.
According to M.R.Hollingdale et al., J.Immunol.132:909-913(1984) method of Miao Shuing is finished the detection method of inhibition sporozoite invasion to be used to estimate the neutralizing effect of the anti-plasmodium falciparum sporozoite of living.In the ISI detection method, at 1%CO
2Use MEM and 10% foetal calf serum with human liver cancer cell cloned cell line Hep G-2-Alb on the glass cover slide
2Be cultured near merging.With substratum dilution antiserum(antisera) or antibody purification (seeing the following form) and be added in the cell culture.30,000 subtertian malaria atom insect spores countings that will be separated to from the mosquito sialisterium of dissecting dilute and are added in each cell culture.At 37 ℃ culture was cultivated 2.5 hours, embathed with PES, and fix with methyl alcohol.In detecting, immunoperoxidase antibody reacts, to observe the sporozoite of invasion with mAb that can discern the proteinic a kind of mark of plasmodium falciparum CS and fixed culture.Then, adopt 400 * the sporozoite number of phase microscope counting invasion.The ISI value is to detect antibody, and hommization antibody exists down, compares the percentage ratio that the invasion spore count reduces with contrast (promptly irrelevant) antibody.This detection method puts in order antibody according to its relative effectivenes.
Following tabulation provides with the previously described chimeric result who obtains when detecting with synthetic antibody.Given value is to suppress percentage ratio and is the mean value of 2-3 independent detection.
The summary of ISI research
In μg/ml: 20 10 5.0 2.0 1.0 0.1
Chimeric antibody 99 98.5 98 88 83 50
PfHzhc2-3/lc1-1 92 75.5 60
PfHzhc2-3/lc1-2 85 80.0 0
PfHzhc2-6/lc1-1 90.0 75 53 0
PfHzhc2-6/lc1-2 87.0 65 50 0
Comprised many modifications and variations form of the present invention in the above in the specification sheets of Miao Shuing, and expected that these modifications and variations all are conspicuous to the arbitrary technician in this area.For example, can provide the recombinant antibodies of the pathogenic agent except that plasmodium falciparum that can neutralize to be used to prepare the preventive that can provide according to instruction of the present invention at the passive immunity of other people disease.Preferably, can discern the engineered antibody of repeat region on other malaria pathogenic agent or be the gratifying parent material that is used to prepare passive immunizing agent of the present invention at maybe can the neutralize engineered antibody in this cycle of parasites in any stage of the engineered antibody in any zone that exists on the plasmodium kind surface in life cycle in any stage.It is believed that the compositions and methods of the invention are carried out above-mentioned modifications and variations all to be contained in the scope of this paper claims.
The sequence catalogue
(1) physical data
(ⅰ) applicant: Smithkline Beecham, Corporation
U.S.Government，Secretary of the Navy
U.S.Government，Secretary of the Army
(ⅱ) denomination of invention: make the people have the new antibodies of the passive immunity of antipathogen infection
(ⅲ) sequence number: 61
(ⅳ) contact address:
(A) address: Howson and Howson
(B) street: Box 457,321 Norristown Road
(C) city: Spring House
(D) state: PA
(E) country: USA
(F) postcode: 19477
(ⅴ) computer-reader form:
(A) media types: Floppy dish
(B) computer: IBM PC compatibility
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Pelease #1.0, Version #1.25
(ⅵ) present application materials:
(A) application number:
(B) applying date:
(C) classification number:
(ⅶ) Yi Qian application materials:
(A) application number: US 07/941,654
(B) applying date: on September 9th, 1992
(ⅷ) proxy/commission merchant's data:
(A) name: Bak, Mary E.
(B) registration number: 31,215
(C) data/file number: SBC P50107-1
(ⅸ) telecommunication data:
(A) phone: (215) 540-9200
(B) fax: (215) 540-5818
(2) data of SEQ ID NO:1:
(ⅰ) sequence signature:
(A) length: 164 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:1
(2) data of SEQ ID NO:2:
(ⅰ) sequence signature:
(A) length: 163 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:2
(2) data of SEQ ID NO:3:
(ⅰ) sequence signature:
(A) length: 339 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..339
(ⅹ ⅰ) sequence description: SEQ ID NO:3
(2) data of SEQ ID NO:4:
(ⅰ) sequence signature:
(A) length: 113 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:4
(2) data of SEQ ID NO:5:
(ⅰ) sequence signature:
(A) length: 339 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..339
(ⅹ ⅰ) sequence description: SEQ ID NO:5
(2) data of SEQ ID NO:6:
(ⅰ) sequence signature:
(A) length: 113 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:6
(2) data of SEQ ID NO:7:
(ⅰ) sequence signature:
(A) length: 339 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..339
(ⅹ ⅰ) sequence description: SEQ ID NO:7
(2) data of SEQ ID NO:8:
(ⅰ) sequence signature:
(A) length: 113 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:8
(2) data of SEQ ID NO:9:
(ⅰ) sequence signature:
(A) length: 354 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..354
(ⅹ ⅰ) sequence description: SEQ ID NO:9
(2) data of SEQ ID NO:10:
(ⅰ) sequence signature:
(A) length: 118 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:10
(2) data of SEQ ID NO:11:
(ⅰ) sequence signature:
(A) length: 389 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..366
(ⅹ ⅰ) sequence description: SEQ ID NO:11
(2) data of SEQ ID NO:12:
(ⅰ) sequence signature:
(A) length: 122 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:12
(2) data of SEQ ID NO:13:
(ⅰ) sequence signature:
(A) length: 389 base pairs
(B) type: nucleic acid
(C) number of share of stock: bifilar
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..366
(ⅹ ⅰ) sequence description: SEQ ID NO:13
(2) data of SEQ ID NO:14:
(ⅰ) sequence signature:
(A) length: 122 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:14
(2) data of SEQ ID NO:15:
(ⅰ) sequence signature:
(A) length: 15 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:15
(2) data of SEQ ID NO:16:
(ⅰ) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:16
Ser Tyr Ala Met Ser
1 5
(2) data of SEQ ID NO:17:
(ⅰ) sequence signature:
(A) length: 51 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:17
(2) data of SEQ ID NO:18:
(ⅰ) sequence signature:
(A) length: 17 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:18
(2) data of SEQ ID NO:19:
(ⅰ) sequence signature:
(A) length: 39 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:19
CTCATCTACT ATGGTTACGA CGGGTATGCT ATGGACTAC 39
(2) data of SEQ ID NO:20:
(ⅰ) sequence signature:
(A) length: 13 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:20
Leu?Ile?Tyr?Tyr?Gly?Tyr?Asp?Gly?Tyr?Ala?Met?Asp?Tyr
1 5 10
(2) data of SEQ ID NO:21:
(ⅰ) sequence signature:
(A) length: 51 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:21
AAGAGCTCTC?AGAGCCTTTT?ATACTCGAGC?AATCAAAAGA?ATTACTTGGC?C 51
(2) data of SEQ ID NO:22:
(ⅰ) sequence signature:
(A) length: 17 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:22
(2) data of SEQ ID NO:23:
(ⅰ) sequence signature:
(A) length: 21 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:23
TGGGCGTCAA CTAGGGAATC T 21
(2) data of SEQ ID NO:24:
(ⅰ) sequence signature:
(A) length: 7 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:24
Trp Ala Ser Thr Arg Glu Ser
1 5
(2) data of SEQ ID NO:25:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:25
CAGCAATATT ATAGCTATCC GCGGACG 27
(2) data of SEQ ID NO:26:
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:26
Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr
1 5
(2) data of SEQ ID NO:27:
(ⅰ) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:27
Pro Asn Ala Asn Pro Asn
1 5
(2) data of SEQ ID NO:28:
(ⅰ) sequence signature:
(A) length: 32 base pairs
(B) type: nucleic acid
(C) number of share of stock: sub-thread
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:28
CCAGATGTAA GCTTCAGCTG ACCCAGTCTC CA 32
(2) data of SEQ ID NO:29:
(ⅰ) sequence signature:
(A) length: 47 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:29
CATCTAGATG GCGCCGCCAC AGTACGTTTG ATCTCCAGCT TGGTCCC?47
(2) data of SEQ ID NO:30:
(ⅰ) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:30
(2) data of SEQ ID NO:31:
(ⅰ) sequence signature:
(A) length: 47 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:31
GCCTGCAGCT AGCTGAGGAG ACGGTGACCG TGGTCCCTTG GCCCCAG?47
(2) data of SEQ ID NO:32:
(ⅰ) sequence signature:
(A) length: 15 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:32
(2) data of SEQ ID NO:33:
(ⅰ) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:33
Ser Tyr Ala Met Ser
1 5
(2) data of SEQ ID NO:34:
(ⅰ) sequence signature:
(A) length: 51 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:34
AAGTCCAGTC?AGAGCCTTTT?ATATAGTAGC?AATCAAAAGA?ATTACTTGGC?C
(2) data of SEQ ID NO:35:
(ⅰ) sequence signature:
(A) length: 17 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:35
(2) data of SEQ ID NO:36:
(ⅰ) sequence signature:
(A) length: 21 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:36
TGGGCATCCA CTAGGGAATC T 21
(2) data of SEQ ID NO:37:
(ⅰ) sequence signature:
(A) length: 7 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:37
Trp Ala Ser Thr Arg Glu Ser
1 5
(2) data of SEQ ID NO:38:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:38
CAGCAATATT ATAGCTATCC TCGGACG 27
(2) data of SEQ ID NO:39:
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:39
Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr
1 5
(2) data of SEQ ID NO:40:
(ⅰ) sequence signature:
(A) length: 21 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:40
TGGGCGTCGA CTAGGGAATC T 21
(2) data of SEQ ID NO:41:
(ⅰ) sequence signature:
(A) length: 7 amino acid
(B) type: amino acid
(D) topological framework: the unknown
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:41
Trp Ala Ser Thr Arg Glu Ser
1 5
(2) data of SEQ ID NO:42:
(ⅰ) sequence signature:
(A) length: 366 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) feature:
(A) title/keyword: CDS
(B) location: 1..366
(ⅹ ⅰ) sequence description: SEQ ID NO:42
(2) data of SEQ ID NO:43:
(ⅰ) sequence signature:
(A) length: 122 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:43
(2) data of SEQ ID NO:44:
(ⅰ) sequence signature:
(A) length: 97 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:44
(2) data of SEQ ID NO:45:
(ⅰ) sequence signature:
(A) length: 164 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:45
(2) data of SEQ ID NO:46:
(ⅰ) sequence signature:
(A) length: 164 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:46
(2) data of SEQ ID NO:47:
(ⅰ) sequence signature:
(A) length: 85 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:47
(2) data of SEQ ID NO:48:
(ⅰ) sequence signature:
(A) length: 102 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:48
(2) data of SEQ ID NO:49:
(ⅰ) sequence signature:
(A) length: 101 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:49
(2) data of SEQ ID NO:50:
(ⅰ) sequence signature:
(A) length: 25 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:50
(2) data of SEQ ID NO:51:
(ⅰ) sequence signature:
(A) length: 28 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:51
CCGCAAGCTT GGGCCCTTGG TACTAGCT 28
(2) data of SEQ ID NO:52:
(ⅰ) sequence signature:
(A) length: 75 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:52
(2) data of SEQ ID NO:53:
(ⅰ) sequence signature:
(A) length: 90 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:53
(2) data of SEQ ID NO:54:
(ⅰ) sequence signature:
(A) length: 93 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:54
(2) data of SEQ ID NO:55:
(ⅰ) sequence signature:
(A) length: 86 base pairs
(B) type: nucleic acid
(C) number of share of stock: sub-thread
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:55
(2) data of SEQ ID NO:56:
(ⅰ) sequence signature:
(A) length: 66 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:56
(2) data of SEQ ID NO:57:
(ⅰ) sequence signature:
(A) length: 78 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:57
(2) data of SEQ ID NO:58:
(ⅰ) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) number of share of stock: sub-thread
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:58
(2) data of SEQ ID NO:59:
(ⅰ) sequence signature:
(A) length: 34 base pairs
(B) type: nucleic acid
(C) number of share of stock: single
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅹ ⅰ) sequence description: SEQ ID NO:59
TGGAAAGCTT GGCGCCGCCA CAGTACGTTT GATC 34
(2) data of SEQ ID NO:60:
(ⅰ) sequence signature:
(A) length: 57 base pairs
(B) type: nucleic acid
(C) number of share of stock: two
(D) topological framework: the unknown
(ⅱ) molecule type: the DNA(genome)
(ⅸ) feature:
(A) title/keyword: CDS
(B) location: 1..57
(ⅹ ⅰ) sequence description: SEQ ID NO:60
(2) data of SEQ ID NO:61:
(ⅰ) sequence signature:
(A) length: 19 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:61
Claims (27)
1, a kind of fusion molecule, this molecule comprise coding have antimalarial protozoon antibody antigen-specific an aminoacid sequence first merge pairing body nucleotide sequence, and made this sequence in framework, merge pairing body nucleotide sequence and be connected through operation with second.
2, molecule according to claim 1, the wherein said first fusion pairing body is a kind of synthetic immune globulin variable region nucleotide sequence, this sequence encoding comprises a complementary determining region that comes from plasmodium antibody, the aminoacid sequence that its fragment or equipotential change or modify.
3, molecule according to claim 2, the wherein said second fusion pairing body is allogenic immunoglobulin variable skeleton district.
4, molecule according to claim 2, wherein said variable region nucleotide sequence is selected from the group of being made up of variable region of heavy chain and variable region of light chain.
5, molecule according to claim 4, they are selected from the group of following material composition:
(a) Fig. 5 heavy chain nucleotide sequence;
(b) heavy chain nucleotide sequence among Fig. 6;
(c) a light chain nucleotide sequence among Fig. 2 or Fig. 3;
(d) a light chain nucleotide sequence among Fig. 3;
(e) and their function fragment.
6, according to the molecule of claim 4, wherein said first merges pairing body nucleotide sequence comprises the group that is selected from following material composition
(a) AGCTATGCCATGAGC;
(b) GAAATTAGTGATGGTGGTAGTTACACCTACTATCCA
GACACTGTGACGGGC;
(c) CTCATCTACTATGGTTACGACGGGTATGCTATGGAC
TAC;
(d) AAGAGCTCTCAGAGCCTTTTATACTCGAGCAATCAA
AAGAATTACTTGGCC;
(e) TGGGCGTCAACTAGGGAATCT;
(f) CAGCAATATTATAGCTATCCGCGGACG;
(g) AGCTATGCCATGTCT;
(h) AAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAA
AGAATTACTTGGCC;
(i) TGGGCATCCACTAGGGAATCT;
(j) CAGCAATATTATAGCTATCCTCGGACG;
(k) TGGGCGTCGACTAGGGAATCT;and an allelic
With and equipotential changes or modify, and have the antigen-specific feature of mouse NFS2, described nucleotide sequence can contain or not contain restriction site and be inserted in required the antibody skeleton district or plasmid vector to impel it.
7, a kind of synthetic immune globulin variable region nucleotide sequence, this sequence encoding comprises the complementary determining region that comes from plasmodium antibody, the aminoacid sequence that its fragment or equipotential change or modify.
8, a kind of fusion rotein, it contains, and come from can be in conjunction with first aminoacid sequence of the plasmodium antibody of the antigenic determinant on the plasmodium kind that is positioned at selection, and described sequence has at the antigen-specific of described antibody and is fused on allogenic second aminoacid sequence.
9, fusion rotein according to claim 8, wherein said first aminoacid sequence comprise and are selected from the aminoacid sequence of being made up of following material:
(a) a kind of variable heavy chain sequence of described antibody;
(b) a kind of variable sequence of light chain of described antibody;
(c) district is measured in the complementation of described antibody; And
(d) function fragment of each sequence from (a) to (c).
10, fusion rotein according to Claim 8, wherein said fusion rotein is selected from the group of being made up of following material
(a) a kind of engineered antibody completely, it has and contains the heavy and light chain of the segmental total length in the variable region that comes from said plasmodium antibody at least;
(b) (a) in Fab or (Fab ') of engineered antibody
2Fragment;
(c) be derived from a kind of dimer of the heavy chain formation of the engineered antibody in (a);
(d) the Fv fragment of the engineered antibody in (a); And
(e) derive from a single-chain antibody of the engineered antibody in (a);
Described protein has the specificity that is same as plasmodium antibody.
11, a kind of plasmodium falciparum engineered antibody, this antibody comprise a heavy chain that contains complementary determining region, and this complementary determining region derives from a kind of variable heavy chain district of inhuman plasmodium falciparum monoclonal antibody.
12, according to the antibody of claim 11, a kind of material in the group that wherein said inhuman CDRs is with following material is formed carries out exercisable the connection
(a) a kind of selected human antibody heavy chain's skeleton and constant region; An and constant region that (b) comes from the heavy chain skeleton of described antibody and come from a kind of selected people's antibody.
13,, further comprise a light chain in the group that is selected from following material composition according to the antibody of claim 11
(a) contain the light chain of the CDR in the variable light chain district that derives from described monoclonal antibody, described variable light chain district carries out exercisable the combination with selected human antibody light chain skeleton and constant region;
(b) constant region that derives from the light chain skeleton of described antibody and derive from selected people's antibody;
(c) the whole light chains that obtain from described antimalarial protozoon antibody; And
(d) come from whole light chains of selected people's antibody.
14, according to the antibody of claim 11, wherein said heavy chain contains and is selected from Fig. 5, the variable heavy chain sequence of Pfhzhc2-3 among Fig. 6 and the sequence of Pfhzhc2-6.
15, according to the antibody of claim 13, wherein said light chain comprises a variable sequence of light chain of the sequence that is selected among Fig. 2 and Fig. 3.
16, according to the antibody of claim 13, light chain complementary determining region wherein is selected from one or more sequences of being made up of following material
(a) LysSerSerGlnSerLeuLeuTyrSerSerAsn
GlnLysAsnTyrLeuAla;
(b) TrpAlaSerThrArgGluSer;and
(c) GlnGlnTyrTyrSerTyrProArgThr.
17, according to the antibody of claim 11, wherein said heavy chain complementary determining region is selected from one or more sequences of being made up of following material
(a) SerTyrAlaMetSer;
(b) GluIleSerAspGlyGlySerTyrThrTyrTyrPro
AspThrValThrGly;and
(c) LeuIleTyrTyrGlyTyrAspGlyTyrAlaMet
AspTyr.
18, a kind of anti-plasmodium falciparum complementary determining region peptide and a kind of analogue thereof that is selected from following group, it has the feature of the antigen-specific of NFS2,
(a) SerTyrAlaMetSer;
(b) GluIleSerAspGlyGlySerTyrThrTyrTyr
ProAspThrValThrGly;
(c) LeuIleTyrTyrGlyTyrAspGlyTyrAlaMet
AspTyr;
(d) LysSerSerGlnSerLeuLeuTyrSerSerAsn
GlnLysAsnTyrLeuAla;
(e) TrpAlaSerThrArgGluSer;
(f) GlnGlnTyrTyrSerTyrProArgThr;
19, a kind of synthetic immunoglobulin variable chain amino acid sequence, this sequence contains the complementary determining region that derives from plasmodium antibody or has the antimalarial protozoon antigen-specific of said sequence in allos variable chains skeleton a fragment or its analogue.
20, according to the sequence of claim 19, it is selected from by Fig. 5, and Fig. 6 is in the group that the aminoacid sequence of Fig. 2 and Fig. 3 is formed.
21, a kind of monoclonal antibody except that NFS2, it has the ability that is attached on the plasmodium falciparum antigenic determinant that contains sequence Pro Asn Ala Asn Pro Asn, or Fab fragment of this antibody or (Fab ')
2Fragment.
22, a kind of chemoprophylaxis composition contains acceptable carrier or thinner on any fusion rotein of claim 8 to 21 or antibody and the pharmacology.
23, pharmaceutical composition according to claim 22, wherein said protein are a kind of hommization plasmodium falciparum antibody.
24, a kind of recombinant plasmid, comprise with one regulate control sequence and carry out operating any one nucleotide sequence of bonded claim 1 to 7, this adjusting control sequence can instruct said nucleotide sequence to duplicate in host cell and express.
25, at least a recombinant plasmid mammalian cells transfected of a kind of usefulness system, this plasmid contains the nucleotide sequence in each of claim 1 to 7.
26, the method for preparation engineering antibody, be included in and be suitable for making under the condition of heavily complementary and light chain expression and assembling, mammal cell line is contained each the recombinant plasmid transfection of nucleotide sequence of claim 1 to 7 with at least a, and from cell culture, reclaim the antibody of assembling.
27, protein or the antibody with claim 8 to 21 is used for pharmaceutical compositions, and said composition is suitable for the infection of passivity ground protection human body opposing plasmodium kind.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US94165492A | 1992-09-09 | 1992-09-09 | |
| US941,654 | 1992-09-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1087681A true CN1087681A (en) | 1994-06-08 |
Family
ID=25476840
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93119080A Pending CN1087681A (en) | 1992-09-09 | 1993-09-09 | New antibodies with passive immunity of pathogenic bacterial infection in the anti-human body |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0659192A4 (en) |
| JP (1) | JPH08500979A (en) |
| KR (1) | KR950703573A (en) |
| CN (1) | CN1087681A (en) |
| AU (1) | AU4851193A (en) |
| CA (1) | CA2143417A1 (en) |
| NZ (1) | NZ256232A (en) |
| WO (1) | WO1994005690A1 (en) |
| ZA (1) | ZA936260B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117854601A (en) * | 2024-03-04 | 2024-04-09 | 鲁东大学 | Decisive complementary region dividing method based on gene type and amino acid sequence |
Families Citing this family (42)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10506274A (en) * | 1994-09-19 | 1998-06-23 | リークスラントボウユニフエルジタイト・ワゲニンゲン | Gene constructs encoding crop protection agents, and transformed plants containing and expressing such constructs, and methods of regulating disease organisms and pathogens in crops |
| SE9500148D0 (en) * | 1995-01-18 | 1995-01-18 | Bioinvent Internatioal Ab | Antibodies for use in cancer therapy and diagnosis |
| AUPP221098A0 (en) | 1998-03-06 | 1998-04-02 | Diatech Pty Ltd | V-like domain binding molecules |
| US6455677B1 (en) | 1998-04-30 | 2002-09-24 | Boehringer Ingelheim International Gmbh | FAPα-specific antibody with improved producibility |
| EP0953639A1 (en) * | 1998-04-30 | 1999-11-03 | Boehringer Ingelheim International GmbH | FAPalpha-specific antibody with improved producibility |
| US20030133939A1 (en) | 2001-01-17 | 2003-07-17 | Genecraft, Inc. | Binding domain-immunoglobulin fusion proteins |
| US7829084B2 (en) | 2001-01-17 | 2010-11-09 | Trubion Pharmaceuticals, Inc. | Binding constructs and methods for use thereof |
| AU2008200400B2 (en) * | 2001-01-17 | 2012-06-07 | Aptevo Research And Development Llc | Binding domain-immunoglobulin fusion proteins |
| RU2420537C2 (en) * | 2001-01-17 | 2011-06-10 | Трабьон Фармасьютикалз Инк. | Fused proteins binding immunoglobulin domain |
| US7754208B2 (en) | 2001-01-17 | 2010-07-13 | Trubion Pharmaceuticals, Inc. | Binding domain-immunoglobulin fusion proteins |
| KR101015496B1 (en) | 2001-01-26 | 2011-02-22 | 인히비텍스, 인코포레이티드 | Monoclonal antibodies to the clfa protein and method of use in treating or preventing infections |
| US8597911B2 (en) | 2003-06-11 | 2013-12-03 | Chugai Seiyaku Kabushiki Kaisha | Process for producing antibodies |
| US7754209B2 (en) | 2003-07-26 | 2010-07-13 | Trubion Pharmaceuticals | Binding constructs and methods for use thereof |
| TW200530269A (en) | 2003-12-12 | 2005-09-16 | Chugai Pharmaceutical Co Ltd | Anti-Mpl antibodies |
| WO2006106905A1 (en) | 2005-03-31 | 2006-10-12 | Chugai Seiyaku Kabushiki Kaisha | Process for production of polypeptide by regulation of assembly |
| US9493569B2 (en) | 2005-03-31 | 2016-11-15 | Chugai Seiyaku Kabushiki Kaisha | Structural isomers of sc(Fv)2 |
| CN101262885B (en) | 2005-06-10 | 2015-04-01 | 中外制药株式会社 | Pharmaceutical compositions containing sc(Fv)2 |
| CA2610987C (en) | 2005-06-10 | 2013-09-10 | Chugai Seiyaku Kabushiki Kaisha | Stabilizer for protein preparation comprising meglumine and use thereof |
| EP1912675B1 (en) | 2005-07-25 | 2014-02-12 | Emergent Product Development Seattle, LLC | B-cell reduction using cd37-specific and cd20-specific binding molecules |
| US11046784B2 (en) | 2006-03-31 | 2021-06-29 | Chugai Seiyaku Kabushiki Kaisha | Methods for controlling blood pharmacokinetics of antibodies |
| EP4218801A3 (en) | 2006-03-31 | 2023-08-23 | Chugai Seiyaku Kabushiki Kaisha | Antibody modification method for purifying bispecific antibody |
| US8409577B2 (en) | 2006-06-12 | 2013-04-02 | Emergent Product Development Seattle, Llc | Single chain multivalent binding proteins with effector function |
| ES2687808T3 (en) | 2007-09-26 | 2018-10-29 | Chugai Seiyaku Kabushiki Kaisha | Constant region of modified antibody |
| SI2202245T1 (en) | 2007-09-26 | 2016-10-28 | Chugai Seiyaku Kabushiki Kaisha | Method of modifying isoelectric point of antibody via amino acid substitution in cdr |
| JP6013733B2 (en) | 2008-04-11 | 2016-10-25 | エマージェント プロダクト デベロップメント シアトル， エルエルシー | CD37 immunotherapeutic and its combination with bifunctional chemotherapeutics |
| JP5717624B2 (en) | 2009-03-19 | 2015-05-13 | 中外製薬株式会社 | Antibody constant region variants |
| TWI544077B (en) | 2009-03-19 | 2016-08-01 | Chugai Pharmaceutical Co Ltd | Antibody constant region change body |
| EP2473530B1 (en) * | 2009-09-03 | 2015-04-22 | Vancouver Biotech Ltd. | Monoclonal antibodies against gonadotropin-releasing hormone receptor |
| EP2481752B1 (en) | 2009-09-24 | 2016-11-09 | Chugai Seiyaku Kabushiki Kaisha | Modified antibody constant regions |
| WO2011108714A1 (en) | 2010-03-04 | 2011-09-09 | 中外製薬株式会社 | Antibody constant region variant |
| KR101398363B1 (en) | 2010-11-17 | 2014-05-22 | 추가이 세이야쿠 가부시키가이샤 | Multi-specific antigen-binding molecule having alternative function to function of blood coagulation factor VIII |
| RU2681885C2 (en) | 2011-10-31 | 2019-03-13 | Чугаи Сейяку Кабусики Кайся | Antigen-binding molecule having regulated conjugation between heavy-chain and light-chain |
| CN105163765A (en) | 2013-03-13 | 2015-12-16 | 伊麦吉纳博公司 | Antigen binding constructs to CD8 |
| EP3050896B1 (en) | 2013-09-27 | 2021-07-07 | Chugai Seiyaku Kabushiki Kaisha | Method for producing polypeptide heteromultimer |
| FR3025517B1 (en) | 2014-09-10 | 2016-12-23 | Repropharm | LIGANDS POTENTIATING THE BIOACTIVITY OF GONADOTROPHINS |
| MA40764A (en) | 2014-09-26 | 2017-08-01 | Chugai Pharmaceutical Co Ltd | THERAPEUTIC AGENT INDUCING CYTOTOXICITY |
| US11142587B2 (en) | 2015-04-01 | 2021-10-12 | Chugai Seiyaku Kabushiki Kaisha | Method for producing polypeptide hetero-oligomer |
| EP4137158A1 (en) | 2015-08-07 | 2023-02-22 | Imaginab, Inc. | Antigen binding constructs to target molecules |
| KR20180053322A (en) | 2015-09-21 | 2018-05-21 | 압테보 리서치 앤드 디벨롭먼트 엘엘씨 | CD3 binding polypeptide |
| MX2018007781A (en) | 2015-12-28 | 2018-09-05 | Chugai Pharmaceutical Co Ltd | Method for promoting efficiency of purification of fc region-containing polypeptide. |
| KR20180116215A (en) | 2016-03-14 | 2018-10-24 | 추가이 세이야쿠 가부시키가이샤 | Cytotoxicity-inducing therapeutic agent for treating cancer |
| US11266745B2 (en) | 2017-02-08 | 2022-03-08 | Imaginab, Inc. | Extension sequences for diabodies |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4632909A (en) * | 1983-10-31 | 1986-12-30 | The United States Of America As Represented By The Department Of Health And Human Services | Monoclonal antibodies which block infectivity of malarial parasites to mosquitoes |
| JPS63267295A (en) * | 1986-12-03 | 1988-11-04 | Sumitomo Chem Co Ltd | Human antibody, antibody gene and corresponding recombinant |
-
1993
- 1993-08-26 ZA ZA936260A patent/ZA936260B/en unknown
- 1993-09-08 AU AU48511/93A patent/AU4851193A/en not_active Abandoned
- 1993-09-08 EP EP93921412A patent/EP0659192A4/en not_active Withdrawn
- 1993-09-08 CA CA002143417A patent/CA2143417A1/en not_active Abandoned
- 1993-09-08 NZ NZ256232A patent/NZ256232A/en unknown
- 1993-09-08 WO PCT/US1993/008435 patent/WO1994005690A1/en not_active Application Discontinuation
- 1993-09-08 JP JP6507519A patent/JPH08500979A/en active Pending
- 1993-09-09 CN CN93119080A patent/CN1087681A/en active Pending
-
1995
- 1995-03-09 KR KR1019950700936A patent/KR950703573A/en not_active Application Discontinuation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117854601A (en) * | 2024-03-04 | 2024-04-09 | 鲁东大学 | Decisive complementary region dividing method based on gene type and amino acid sequence |
| CN117854601B (en) * | 2024-03-04 | 2024-05-14 | 鲁东大学 | Decisive complementary region dividing method based on gene type and amino acid sequence |
Also Published As
| Publication number | Publication date |
|---|---|
| NZ256232A (en) | 1997-02-24 |
| AU4851193A (en) | 1994-03-29 |
| WO1994005690A1 (en) | 1994-03-17 |
| CA2143417A1 (en) | 1994-03-17 |
| JPH08500979A (en) | 1996-02-06 |
| ZA936260B (en) | 1994-03-18 |
| EP0659192A1 (en) | 1995-06-28 |
| KR950703573A (en) | 1995-09-20 |
| EP0659192A4 (en) | 1996-09-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1087681A (en) | New antibodies with passive immunity of pathogenic bacterial infection in the anti-human body | |
| CN1202128C (en) | Method for reducing immunogenicity of proteins | |
| CN1246335C (en) | Recombinant IL-18 antagonist useful in treatment of IL-18 mediated disorders | |
| CN1207306C (en) | Peptide compound for preventing and treating HIV infection and immunity diseases | |
| CN1211123C (en) | Humanized antibodies against leukocyte adhesion molecule VLA-4 | |
| CN1187374C (en) | Humanized antibody specific for human 4-1BB and pharmaceutical compsn comprising same | |
| CN1174998C (en) | Peptide immunogens for vaccination against and treatment of allergy | |
| CN1271205C (en) | Reconstituted human anti-HM1.24 antibody | |
| CN1076966A (en) | The clone of the humanized monoclonal antibodies of anti-human interleukin-4 and expression | |
| CN1163599C (en) | anti-GPIIb/IIIa recombinant antibodies | |
| CN1105728C (en) | Recombinant IL4 antibodies useful in treatment of IL4 mediated disorders | |
| CN1077991A (en) | The design of monoclonal antibody with anti-Ro 24-7472/000-5 of people's attribute, clone and expressing | |
| CN1745100A (en) | Factor VIII polypeptide | |
| CN1273588A (en) | Amino acid sequences for therapeutical and prophylacticapplications to disedses due to i (clostridium difficile) toxinc | |
| CN1884303A (en) | SARS-CoV virus structural protein infusion protein and its high yield expression and purification and uses | |
| CN1279690A (en) | Soluble single-chain T-cell receptor proteins | |
| CN1200737A (en) | Recombinant anti-CD4 antibodies for human therapy | |
| CN1558916A (en) | Compositions and methods for generating chimeric heteromultimers | |
| CN1520423A (en) | Epitopes or mimotopes derived from C-eqsilon-3, or C-epsilon-4 domains of ige, antagonists thereof, and their thera peutic uses | |
| CN1688338A (en) | Method of humanizing immune system molecules | |
| CN1526072A (en) | Method for the identification of extracellular domains of the epstein barr virus (ebv), tumor-associated latent membrane proteins and for the selection of antibody reagents reactive therewith | |
| CN1078250C (en) | Reshaped monoclonal antibodies against an immunoglobulin isotype | |
| CN1227610A (en) | Antibodies against a complex of CD4 and a chemokine receptor domain, and their use against HIV infections | |
| CN87105411A (en) | The T cell in S district and B cell antigen decision base before the hepatitis B virus surface antigen | |
| CN1531548A (en) | Chimeric chains that code for proteins that induce effects directed against viruses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |