CN1272345C - Silensed anti-CD 28 antibodies and use thereof - Google Patents
Silensed anti-CD 28 antibodies and use thereof Download PDFInfo
- Publication number
- CN1272345C CN1272345C CNB018226361A CN01822636A CN1272345C CN 1272345 C CN1272345 C CN 1272345C CN B018226361 A CNB018226361 A CN B018226361A CN 01822636 A CN01822636 A CN 01822636A CN 1272345 C CN1272345 C CN 1272345C
- Authority
- CN
- China
- Prior art keywords
- antibody
- ser
- cell
- leu
- gly
- 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.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 claims abstract description 53
- 239000000203 mixture Substances 0.000 claims abstract description 20
- 210000000056 organ Anatomy 0.000 claims abstract description 8
- 230000014509 gene expression Effects 0.000 claims description 32
- 239000013604 expression vector Substances 0.000 claims description 25
- 108091033319 polynucleotide Proteins 0.000 claims description 13
- 102000040430 polynucleotide Human genes 0.000 claims description 13
- 239000002157 polynucleotide Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 239000003018 immunosuppressive agent Substances 0.000 claims description 4
- 229940124589 immunosuppressive drug Drugs 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 6
- 238000002054 transplantation Methods 0.000 claims 1
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 29
- 206010052779 Transplant rejections Diseases 0.000 abstract description 8
- 230000002297 mitogenic effect Effects 0.000 abstract description 7
- 230000001506 immunosuppresive effect Effects 0.000 abstract description 4
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 206010062016 Immunosuppression Diseases 0.000 abstract description 2
- 230000002950 deficient Effects 0.000 abstract 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 150
- 210000004027 cell Anatomy 0.000 description 147
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical group OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 75
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 59
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 59
- 241000699666 Mus <mouse, genus> Species 0.000 description 57
- 108090000623 proteins and genes Proteins 0.000 description 35
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 32
- 150000001413 amino acids Chemical class 0.000 description 29
- 108020004414 DNA Proteins 0.000 description 27
- 238000012875 competitive assay Methods 0.000 description 21
- 239000002299 complementary DNA Substances 0.000 description 21
- 238000012360 testing method Methods 0.000 description 21
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 18
- 238000002965 ELISA Methods 0.000 description 17
- 238000004043 dyeing Methods 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 16
- 238000003752 polymerase chain reaction Methods 0.000 description 15
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 15
- 241000283707 Capra Species 0.000 description 14
- 241000699800 Cricetinae Species 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 238000003146 transient transfection Methods 0.000 description 14
- 210000004408 hybridoma Anatomy 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 238000000684 flow cytometry Methods 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- 230000002829 reductive effect Effects 0.000 description 12
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 241000283074 Equus asinus Species 0.000 description 9
- 230000027455 binding Effects 0.000 description 9
- 230000008859 change Effects 0.000 description 9
- 238000001890 transfection Methods 0.000 description 9
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 8
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 8
- 229940045513 CTLA4 antagonist Drugs 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 210000000612 antigen-presenting cell Anatomy 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000013207 serial dilution Methods 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 230000004913 activation Effects 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 239000013613 expression plasmid Substances 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 210000004698 lymphocyte Anatomy 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000003153 stable transfection Methods 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 230000009871 nonspecific binding Effects 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 102220023256 rs387907547 Human genes 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 239000011735 vitamin B7 Substances 0.000 description 5
- 102000010910 CD28 Antigens Human genes 0.000 description 4
- 108010062433 CD28 Antigens Proteins 0.000 description 4
- 102100032937 CD40 ligand Human genes 0.000 description 4
- 241000699679 Cricetulus migratorius Species 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 230000006044 T cell activation Effects 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 102220369445 c.668T>C Human genes 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 3
- 108700031361 Brachyury Proteins 0.000 description 3
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 3
- 102000004594 DNA Polymerase I Human genes 0.000 description 3
- 108010017826 DNA Polymerase I Proteins 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 3
- 206010003246 arthritis Diseases 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000004040 coloring Methods 0.000 description 3
- 238000010612 desalination reaction Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 101150106093 gpt gene Proteins 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000009413 insulation Methods 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 description 3
- 229940093916 potassium phosphate Drugs 0.000 description 3
- 235000011009 potassium phosphates Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000012460 protein solution Substances 0.000 description 3
- 238000013094 purity test Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000004988 splenocyte Anatomy 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 238000010361 transduction Methods 0.000 description 3
- 230000026683 transduction Effects 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 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 2
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 2
- 102000004400 Aminopeptidases Human genes 0.000 description 2
- 108090000915 Aminopeptidases Proteins 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 238000011735 C3H mouse Methods 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 102000018361 Contactin Human genes 0.000 description 2
- 108060003955 Contactin Proteins 0.000 description 2
- 101150074155 DHFR gene Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108090000270 Ficain Proteins 0.000 description 2
- 101000840258 Homo sapiens Immunoglobulin J chain Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108090001090 Lectins Proteins 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 108010029180 Sialic Acid Binding Ig-like Lectin 3 Proteins 0.000 description 2
- 102000001555 Sialic Acid Binding Ig-like Lectin 3 Human genes 0.000 description 2
- 108700007696 Tetrahydrofolate Dehydrogenase Proteins 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 108010027570 Xanthine phosphoribosyltransferase Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000002917 arthritic effect Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 102220369446 c.1274G>A Human genes 0.000 description 2
- 102220369447 c.1352G>A Human genes 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 238000005094 computer simulation Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000002249 digestive system Anatomy 0.000 description 2
- 102000004419 dihydrofolate reductase Human genes 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- -1 external pulvis Substances 0.000 description 2
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 description 2
- 235000019836 ficin Nutrition 0.000 description 2
- 239000012997 ficoll-paque Substances 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 2
- 239000011544 gradient gel Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 230000007794 irritation Effects 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000002073 mitogenetic effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000004976 peripheral blood cell Anatomy 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 229940079889 pyrrolidonecarboxylic acid Drugs 0.000 description 2
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 102220023258 rs387907548 Human genes 0.000 description 2
- 229960002930 sirolimus Drugs 0.000 description 2
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 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
- 206010001497 Agitation Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 101000852101 Bombyx mori DNA topoisomerase 2 Proteins 0.000 description 1
- 102000004497 CCR2 Receptors Human genes 0.000 description 1
- 108010017312 CCR2 Receptors Proteins 0.000 description 1
- 108010029697 CD40 Ligand Proteins 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- UNPLRYRWJLTVAE-UHFFFAOYSA-N Cloperastine hydrochloride Chemical compound Cl.C1=CC(Cl)=CC=C1C(C=1C=CC=CC=1)OCCN1CCCCC1 UNPLRYRWJLTVAE-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 102100026973 Heat shock protein 75 kDa, mitochondrial Human genes 0.000 description 1
- 101100166600 Homo sapiens CD28 gene Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102100022723 Interleukin-22 receptor subunit alpha-1 Human genes 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- ZOKVLMBYDSIDKG-CSMHCCOUSA-N Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN ZOKVLMBYDSIDKG-CSMHCCOUSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100370002 Mus musculus Tnfsf14 gene Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- ROHDXJUFQVRDAV-UWVGGRQHSA-N Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 ROHDXJUFQVRDAV-UWVGGRQHSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 1
- 101710204707 Transforming growth factor-beta receptor-associated protein 1 Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000003172 anti-dna Effects 0.000 description 1
- 230000007503 antigenic stimulation Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 239000003885 eye ointment Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 229960002706 gusperimus Drugs 0.000 description 1
- IDINUJSAMVOPCM-UHFFFAOYSA-N gusperimus Chemical compound NCCCNCCCCNC(=O)C(O)NC(=O)CCCCCCN=C(N)N IDINUJSAMVOPCM-UHFFFAOYSA-N 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 108010027445 interleukin-22 receptor Proteins 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000023404 leukocyte cell-cell adhesion Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 125000003071 maltose group Chemical group 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000010363 phase shift Effects 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229940072288 prograf Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000001846 repelling effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 101150112970 up gene Proteins 0.000 description 1
- 239000006216 vaginal suppository Substances 0.000 description 1
- 229940120293 vaginal suppository Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 108700026215 vpr Genes Proteins 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Transplantation (AREA)
- Surgery (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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides anti-CD28 antibodies which are defective in mitogenic activity (silenced anti-CD28 antibodies), methods of producing, compositions containing the antibody and methods of immunosuppression, inducing T-cell tolerance and treating organ and/or tissue transplant rejections.
Description
Invention field
The present invention relates to lack the anti-CD-28 antibody and the application thereof of mitogenic activity.
Background of invention
The transplant rejection of immune response, especially organ mainly results from the lymphocytic activation of T.This activation of T cell is by a kind of signal induction from antigen presenting cell (APC).This signal from APC relates to first signal by TXi Baoshouti (TCR), relates to second signal (common stimulus signal) by co stimulatory molecule.When APC presented the T cell antigen by TCR, described first signal was from the antigenic major histocompatibility antigen of peptide (MHC) complex body.Described second signal is mediated by several co stimulatory molecules; The example of described co stimulatory molecule comprises: APC one side be called the B7 (B7-1 (CD80) and B7-2 (CD86)) of part and in T cell one side as CD28, CTLA-4 of acceptor or the like.Part B7 is a kind of glycoprotein of contactin, and expresses in the B cell that belongs to the antigen presenting cell monoid etc.Identification all is the transmembrane glycoprotein of contactin as CD28 and the CTLA-4 of the B7 of common ligands.Therefore, by transduceing simultaneously, regulate the activation of T cell via first signal of TCR with from the second signal of for example B7 and CD28/CTLA-4.Know that the signal from B7 to CD28 promotes the activation of T cell, the then activation of suppressor T cell of the signal from B7 to CTLA-4 [Waterhouse etc., Science, 270:985-988 (1995)].
Till now, for induction of immunity suppresses or tolerance, attempted waiting and blocking the B7-CD28 signal by giving CTLA-4Ig, anti-B7-1 antibody/anti-B7-2 antibody, anti-CD28 antibody.For example, CTLA-4Ig is in conjunction with B7, thus the reaction between interference B7 and the CD28, and therefore blocking-up demonstrates immunosuppressive activity from the signal of CD28.But, because the reaction between B7 and the CTLA-4 also is suppressed simultaneously, thereby also suppresses the T cell activation is risen the CTLA-4 signal of down regulation, so that can't bring out required tolerance (Kirk etc., Proc.Natl.Acad.Sci.USA, 94:8789-8794 (1997)).Prepared anti-B7 antibody in addition, it is reported the only suppressor T cell activation under the CTLA-4Ig situation of anti-B7 antibody, this antibody also suppresses the CTLA-4 signal.In experiment in vitro, find that anti-CD28 antibody produces the mitogenesis effect to the T cell, and then promote the growth and the activation of T cell with the combined stimulation of this antibody and anti-cd 3 antibodies, and [WO 90/05541 to increase the output of cytokine, Eur.J.Immunology, 16,1289-1296 (1986) or the like].In addition, inspire of the mitogenesis stimulation of anti-CD28 antibody in vivo, cause being similar to the generation [Yin etc., J.Immunology, 163:4328-4334 (1999)] of the T cell activation signal of the second signal from B7 to CD28 T cell CD28 acceptor.These T cell activation functions then hint: can will resist CD28 antibody as immunostimulant (WO 90/05541) in cancer and acquired immune deficiency syndrome (AIDS) (AIDS) treatment.
Summary of the invention
Anti-CD28 antibody with the routine techniques preparation produces mitogenetic effect to the T cell.Though also not exclusively understand the reason of this mitogenic activity, it is believed that the Fc district of anti-CD28 is possible reason (Cole etc., J.Immunology, 36:159 (1997)) with combining of antigen presenting cell Fc acceptor.Therefore, we are by using genetic engineering technique, sudden change are incorporated in the binding site of Fc acceptor of anti-CD28 antibody, thereby modify this antibody, make it can not have mitogenic activity again.The inventor has prepared a kind of such antibody-TN228IgG2M3, and in this antibody, IgG2M3 has two amino acid whose replacements in the IgG gene.And we have confirmed that the anti-CD28 antibody of the reticent type that is produced does not have mitogenetic activity, and this is very useful for inducing T cell tolerance.
Therefore, the invention provides the anti-CD28 antibody (being referred to as the anti-CD28 antibody of reticent type hereinafter) that does not have mitogenic activity, and provide by suppressing immune response (especially transplant rejection) with described antibody and bringing out the method for immunological tolerance.
A target of the present invention is the anti-CD28 antibody of reticent type, and wherein said anti-CD28 antibody can be chimeric antibody and/or humanized antibody.The variable region of described anti-CD28 antibody can comprise with the aminoacid sequence shown in the SEQ ID NO:2,4,7 and 9 and the polynucleotide of such aminoacid sequence of encoding.For example, such polynucleotide comprise SEQ ID NO:1,3,6 and 8.
Another target of the present invention is: the carrier and the cell host that comprise the polynucleotide of the described anti-CD28 antibody of encoding.
Another target of the present invention is: by under the condition that allows described polynucleotide to express, cultivate the cell host of the polynucleotide that comprise the anti-CD28 antibody of encoding and this gene product that collection produced, the method for producing the anti-CD28 antibody of reticent type.
Another target of the present invention is to comprise the medicinal compositions that one or more plant the anti-CD28 antibody of reticent type, preferably plants the described medicinal compositions of pharmaceutically acceptable composition blended with one or more.
The anti-CD28 antibody of described reticent type can be used for bringing out T cell tolerance, immunosuppression, and can be used as a kind of prevention/remedies of organ or tissue's transplant rejection.Therefore, the invention provides: by give Mammals one or more plant the anti-CD28 antibody of reticent type and bring out T cell tolerance, immunosuppressant method, and during organ or tissue's transplant rejection by give Mammals one or more plant the method that the anti-CD28 antibody of reticent type reaches preventative or therapeutic treatment.Preferably give so reticent type anti-CD28 antibody, and can comprise suitable additional medicine/medicine with medicinal compositions form described herein.
The accompanying drawing summary
Fig. 1: express the plasmid construction body that ChTN228 antibody is used.The VL of mouse TN228 and VH are built into little exon in abutting connection with the XbaI site.Described VL sequence is inserted among the expression vector pVk, and described VH sequence is inserted among the expression vector pVg2M3.
Fig. 2: the nucleotide sequence of ChTN228 light chain and the aminoacid sequence of inferring in the little exon.Signal peptide sequence is represented with italic.The following underlining of CDR.Sophisticated light chain begins with asparagicacid residue (bold-type letter).Non-translated sequence and intron sequences are represented with lowercase.(SEQ ID NO:1 and 2).
Fig. 3: the nucleotide sequence of ChTN228 variable region of heavy chain and the aminoacid sequence of inferring in the little exon.Signal peptide sequence is represented with italic.The following underlining of CDR.Sophisticated heavy chain begins with glutamine residue (bold-type letter).Non-translated sequence and intron sequences are represented with lowercase.(SEQ ID NO:3 and 4).
Fig. 4: competitive assay.With P815/CD28
+Cell is with 2 times of serial dilution incubation P815/CD28 of 25ng MuTN228-FITC and described or ChTN228 or MuTN228
+Equally, P815/CD28 ' cell with independent MuTN228-FITC incubation, is not added any competitor.The passage fluorescence mean vol of each sample is mapped to the concentration of competitor.
Fig. 5: TN228-IgG2m3 is to the restraining effect of the first MLR of people (1).Shown percent inhibition respectively from the first MLR of four individualities.
Fig. 6: TN228-IgG2m3 is to the restraining effect of the first MLR of people (2).Shown percent inhibition respectively from the first MLR of four individualities.
Fig. 7: TN228-IgG2m3 is to the effect of secondary MLR.Data have been shown respectively from two volunteers.Stimulate as 100 with Raji independent among the first MLR, the form of the dpm percentage ratio that stimulates with independent Raji is represented the absorption of [the 3H]-thymidine among the secondary MLR.TN228-IgG2m3：0.1mg/ml。
Fig. 8: express the plasmid construction body that HuTN228 antibody is used.The VL of humanization TN228 and VH are built into little exon in abutting connection with the XbaI site.Described VL sequence is inserted among the expression vector pVk, and described VH sequence is inserted among the expression vector pVg2M3.
Fig. 9: the nucleotide sequence of HuTN228 variable region of heavy chain and the aminoacid sequence of inferring in the little exon.Signal peptide sequence is represented with italic.The following underlining of CDR.Sophisticated heavy chain begins with glutamine residue (bold-type letter).(SEQ ID NO:6 and 7).
Figure 10: the nucleotide sequence of HuTN228 variable region of light chain and the aminoacid sequence of inferring in the little exon.Signal peptide sequence is represented with italic.The following underlining of CDR.Sophisticated light chain begins with asparagicacid residue (bold-type letter).(SEQ ID NO:8 and 9).
Figure 11: FACS competitive assay.With the flow cytometer competitive assay of describing in an embodiment, the MuTN228 that has analyzed the FITC mark under the situation that has different amount competitor MuTN228 antibody or HuTN228 antibody with the combining of P815/CD28 ' cell.
Figure 12: ELISA competitive assay.With the ELISA competitive assay of describing in an embodiment, analyzed biotinylated MuTN228 under the situation that has different amount competitor MuTN228 antibody or HuTN228 antibody with the combining of sCD28-Fc.
Figure 13: I-125 competitive assay.With what describe in an embodiment
125I traget antibody competitive assay has been analyzed
125The MuTN228 of I mark under the situation that has different amount competitor MuTN228 antibody or HuTN228 antibody with P815/CD28
+The combination of cell.
Figure 14: express the plasmid construction body that PV1-IgG3 antibody is used.V with PV1
LAnd V
HBe built into little exon in abutting connection with the XbaI site.Described V
LSequence is inserted among the expression vector pMVk.rg.dE, and with described V
HSequence is inserted among the expression vector pMVg3.D.Tt.Then, the described two kinds of plasmids of recombinating, the single plasmid of generation coexpression PV1-IgG3 heavy chain and PV1-IgG3 light chain.
Figure 15 A: the cDNA sequence of light chain and heavy chain described in the little exon and the aminoacid sequence of inferring.The following underlining of CDR.Asparagicacid residue (the double underline mark) beginning of sophisticated light chain to be positioned at position 20.(SEQ ID NO:10 and 11).
Figure 15 B: the cDNA sequence of the PV1 variable region in the little exon and the aminoacid sequence of inferring.The following underlining of CDR.Glutamine (the double underline mark) beginning of sophisticated heavy chain to be positioned at position 20.(SEQ ID NO:12 and 13).
Figure 16: according to what describe in the method, utilization HPLC, the PV-1-IgG3 that is undertaken by size exclusion chromatography analyzes.Monitor albumen according to albumen in the absorbancy of 280nM.
Figure 17: the SDS-PAGE of the contrast of mouse IgG3 isotype (swimming lane 1), PV1 (swimming lane 2) and PV1-IgG3 (swimming lane 3) analyzes.Albumen in the A plate is electrophoresis under non-reduced condition, and the electrophoresis under reductive condition of the albumen in the B plate.MW represents molecular weight marker.Institute's column of figure is to be the MW standard of unit with kD.
Figure 18: with PV1 (A), 37.51 (B) or PV1-IgG3 (C) is painted and with the EL4 cell of flow cytometry analysis.Used second antibody is: is used for the anti-armenian hamster IgG of the donkey of puting together FITC (H+L) of PV1, is used for 37.51 the anti-gold hamster IgG of the donkey of puting together FITC, and the goat anti-mouse κ that puts together FITC that is used for PV1-IgG3.The distribution plan of solid line is represented only by the painted cell of second antibody.The dotted line distribution plan is represented first antibody and the painted cell of second antibody described in the method.The contrast of mouse IgG3 isotype does not make EL4 cell dyeing (data not shown).
Figure 19: (A) excessive PV1 or PV1-IgG3 with put together the PV1 competition of R-PE and combining of EL4 cell.The cell of any look is not dyed in fine line in the flow cytometry histogram (black) expression, heavy line (mazarine) expression is only by the painted cell of R-PE-PV1, fine dotted line (carmetta) expression is by R-PE-PV1 and the painted cell of excessive unconjugated PV1, and thin doublet (light blue) expression is by R-PE-PV1 and the painted cell of excessive unconjugated PV1-IgG3.Excessive mouse IgG3 isotype contrast is to combine the not influence (data not shown) of R-PE-PV1 with the EL4 cell.(B) excessive 145.2C11 or the 145.2C11-IgG3 and the 145.2C11 competition of puting together R-PE are attached on the EL4 cell.The no any painted cell of fine line (black) expression, heavy line (mazarine) expression is only by the painted cell of R-PE-145.2C11, fine dotted line (carmetta) expression is by R-PE-145.2C11 and the painted cell of excessive unconjugated 145.2C11, and thin doublet (light blue) expression is by R-PE-145.2C11 and the painted cell of excessive unconjugated 145.2C11-IgG3.(C) excessive PV1 combines the EL4 cell with the PV1-IgG3 competition., or do not having under the situation of excessive PV1 with PV1-IgG3 the EL4 cell dyeing with PV1-IgG3 and excessive PV1 the EL4 cell dyeing.Washed cell, and the anti-mouse IgG of donkey specific with mouse IgG3, that put together FITC (H+L) is with its dyeing.Fine line (black) expression is only by the painted cell of second antibody, and heavy line (mazarine) is represented by PV1-IgG3 and the painted cell of second antibody, and fine dotted line (carmetta) expression is by PV1-IgG3 and excessive PV1 and the painted cell of second antibody.
Figure 20: with PV1-IgG3 and the painted mice spleen cell of 145.2C11.With cell dyeing, redye cell with mouse IgG3 isotype contrast (A) or PV1-IgG3 (B) with goat anti-mouse IgG of puting together R-PE and the 145.2C11 that puts together FITC, and with the bi-color flow cytometry analysis of cells of describing in the materials and methods.Only analyze the cell in the lymphocyte gate.In the PV1-IgG3 positive cell superincumbent " quadrant ", and in CD-3 positive cell " quadrant " on the right.The percentage ratio of the cell of the numeral in each " quadrant " in that specific " quadrant ".
Detailed Description Of The Invention
Within the scope of the present invention, term " the anti-CD28 antibody of reticent type " means any anti-CD28 antibody that lacks mitogenic activity.More precisely, it is a specificity in conjunction with the antigens c D28 acceptor on the T cell surface and can be by not promoting growth of T cell or activatory antibody with the anti-cd 3 antibodies combined stimulation.
Can be on the basis of anti-CD28 antibody or producing on the basis of hybridoma of anti-CD28 antibody, by with genetic engineering technique or by chemically modified, make anti-CD28 antibody mutation or, make up the anti-CD28 antibody of reticent type its modification with excitability.Utilization with genetic engineering technique is an example, by sudden change being incorporated in the aminoacid sequence in described antibody Fc district, can reduce or eliminate the binding affinity of anti-CD28 antibody to the Fc acceptor.For example, by from the hybridoma that can produce anti-CD28 monoclonal antibody, separating cDNA, and with sudden change be incorporated into the corresponding sequence area in the Fc that in combining Fc acceptor process, plays an important role zone in, then can obtain the anti-CD28 antibody of reticent type (WO 88/07089).For the site and without particular limitation of sudden change, because can suppress and the combining of Fc acceptor.Therefore, under the situation of IgG antibody-like, for example H-chain amino acid residue 234,235,236,237,318,320 and 322 is preferred, and can be by replace at least a anti-CD28 antibody of reticent type that makes up in these amino acid with different amino acid.
Can select the source of so anti-CD28 antibody of reticent type carefully according to the target animals of using described antibody.For example, inhuman monoclonal antibody contains and show antigenic aminoacid sequence in the mankind in quite wide in range scope.Many researchs show: after injection of antibodies, the patient is very strong to the immunne response of external antibody; And only give this antibody, may make the patient maybe can make this antibody lose treatment effectiveness among entering precarious position.Therefore, recommendablely be: replace Fc district so that make described antibody and the relative more homology of treatment target animals, replace the frame section of variable region, or use is from the described antibody of the transgenic animal acquisition of having introduced described antibody gene.For example, when will be described antibody administration of human, then available following antibody: by replacing the obtainable chimeric antibody in Fc district (EP125023), the substituted humanized antibody (EP0239400 of frame section, EP045126) or the people's antibody (EP546073, WO 97/07671) that obtains from the transgenic animal of having introduced described human immunoglobulin gene.By in these antibody, introducing sudden change, or in these antibody, introduce sudden change, can reduce or eliminate the mitogenic activity of described antibody by chemically modified with for example above-mentioned those technology of genetic engineering technique.
Specific examples as anti-CD28 antibody with reticent type Fc district, not only can mention the antibody of describing in the part of embodiment hereinafter, and can mention with the constant region gene of the target animals of treat with based on SEQ ID NO:2 and 4 or SEQ ID NO:7 and 9 shown in the variable region polynucleotide of variable region amino acid sequence synthesize the antibody for preparing.The example of such polynucleotide is SEQ ID NO:1,3,6 and 8.
Example more specifically of the present invention be HuTN228 and MuTN228 with and Fab fragment, its F (ab) ' 2 fragment, its derivative or the like.
Expect that as those skilled in the art because the degeneracy of the 3rd base, almost every seed amino acid can be represented by a more than triplet codon in nucleotide coding sequence.And less base pair changes the variation (the conservative replacement) that may cause coded aminoacid sequence; But estimate can significantly not change the biologic activity of described gene product.Therefore, on sequence, can modify the nucleotide sequence (for example replacing a Nucleotide in the triplet codon) of coding albumen disclosed herein or peptide slightly, the gene product of its corresponding, same aminoacid sequence but this sequence is still encoded.
Term " expression vector " be meant the peptide of code book invention and be provided in the selected host cell express this peptide the polynucleotide of essential sequence.Expression vector can comprise transcripting promoter and terminator usually, perhaps will create conditions for contiguous endogenesis promoter mixes.Expression vector can be the plasmid that further comprises a replication orgin and one or more selective marker usually.Yet on the other hand, expression vector can be the viral recon that is designed infection host, or is designed for the integrating vector that a preferred sites in host genome is integrated.At Molecular Cloning:A Laboratory Manual, Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press in 1989, discloses the example of expression vector.
The suitable host cell that is used for the anti-CD28 antibody of expression silencing type comprises: prokaryotic organism, yeast, archeobacteria (archae) and other eukaryotic cell.The suitable cloning vector and the expression vector that use for bacterium, fungi, yeast and mammalian cell host are that the present technique field is well-known, for example, Pouwels etc., Cloning Vectors:A Laboratory Manual, Elsevier, New York (1985).Described cell is mammalian cell preferably.Described carrier can be plasmid vector, strand or double stranded phage carrier or strand or double-stranded RNA or dna viral vector.Can import to technology in the cell to DNA and RNA with well-known,, and, such carrier is incorporated in the cell preferably with the form of DNA with the form of polynucleotide.Under the situation of phage and virus vector, with well-known infection and transduction technology, also can and preferably with the form of packaging virus or envelope virus, described carrier is incorporated in the cell.Virus vector can be have a replication or replication defect type.Under latter event, viral proliferation will only betide in the complementary host cell usually.Also can use the RNA that derives from DNA construct of the present invention, produce described albumen with cell free translation system.
Can come the anti-CD28 antibody/albumen of the reticent type of purifying by the method for common known protein separation/purification in the protein chemistry field.More particularly, for example can mention: extract recrystallization, with saltouing of ammonium sulfate, sodium sulfate or the like, centrifugal, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal-phase chromatography, reversed phase chromatography, gel filtration method, gel permeation chromatography, affinity chromatography, electrophoresis, the combination of adverse current distribution or the like and these methods.
According to the present invention, can come the production antibody purified with above-described recombinant expression system.This method comprises: be enough to promote under the condition of described protein expression the cultivation expression vector transformed host cells of the dna sequence dna that contains this albumen of encoding.Then, decide, from substratum or cell extract, reclaim this albumen according to used expression system.As known to the technician, the step of purification of recombinant proteins, the factor that will whether be secreted in the substratum according to type and this recombinant protein such as used host cell changes.
If the anti-CD28 antibody of reticent type is mixed with a kind of medicinal compositions, then can use it for: the transplant rejection after (a) organ or tissue transplants, described organ or tissue is the heart, kidney, liver, marrow, skin, cornea, lung, pancreas, small intestine, muscle, nerve or the like for example; (b) graft-vs-host reaction during bone marrow transplantation; (c) autoimmune disease of rheumatoid arthritis, systemic lupus erythematous, multiple sclerosis, myasthenia gravis, type i diabetes or the like for example; And (d) Immunological diseases of asthma, atopic dermatitis or the like for example.
Suppress immune response and transplant rejection and bring out immunological tolerance though can estimate the anti-CD28 antibody of reticent type itself, also can unite use with other medicines.Such can be used for the antibody combined other medicines of the anti-CD28 of reticent type among, various immunosuppressive drugs are arranged, for example the antibody and the MMF of rapamycin, Gusperimus, anti-cd40 antibody, anti-CD40L antibodies, Prograf, cyclosporin A, anti-IL-2 antibody, anti-IL-2 acceptor.Particularly rapamycin suppresses from the transduction that relates to T cell growth signals in the signal of IL-2 acceptor, and does not suppress the transduction of apoptosis coherent signal; Therefore expect that the coupling of specific inhibitor of it and CD28 signal will be useful.
Can give reticent type of the present invention anti-CD28 antibody by oral or non-enteron aisle, preferably give by intravenous route, intramuscular approach or subcutaneous route.
Can prepare the anti-CD28 antibody of reticent type of the present invention with the form of solution or lyophilized powder; And in case of necessity, can prepare the anti-CD28 antibody of reticent type of the present invention with pharmaceutically acceptable various additives for example vehicle, thinner, stablizer, isotonic agent and buffer reagent.Preferred additives comprises: sugar is maltose, tensio-active agent polysorbate, amino acid glycine, protein human serum albumin and salt sodium-chlor for example for example for example for example for example.
Equally, can suitably select formulation according to administering mode, described formulation is injection (solution system, suspensoid, emulsion, dissolved solid or the like) in use, tablet, capsule, granule, powder, liquid preparation, liposome inclusion, ointment, gelifying agent, external pulvis, sprays, suction pulvis, eye drops, Eye ointments, suppository, vaginal suppository or the like for example; And can correspondingly prepare peptide of the present invention.At Comprehensive Medicinal Chemistry, the 5th volume, editors such as Hansch in 25.2 chapters and sections of Pergamon Press 1990, have described preparation comprehensively.
The dosage of medicinal compositions of the present invention depends on that especially concrete composition, conduct are treated or type, administering mode, patient's age and the healthy state of the disease of prevention target and the time length of treatment.Yet, give at intravenously, intramuscular gives or subcutaneous situation about giving under, can give every day each adult 0.01-100mg/kg, preferably give 0.1-10mg/kg.
When using the anti-CD28 antibody of reticent type of the present invention to suppress transplant rejection or bringing out immunological tolerance, after organ or tissue transplants, can be by intravenous injection, intramuscularly or subcutaneous injection, dosage with about 1mg/kg/ days, face transplant before, after the transplanting just, and, give described composition transplanting back 3 days, 7 days, 12 days, 18 days, 25 days, 35 days, 45 days and 60 days.In the rejection process after monitoring is transplanted, can increase or reduce the frequency and the dosage of administration carefully.
Though used administering mode and patient's illness is especially depended at the interval of administration; But not only successive administration but also intermittently administration all are feasible.So, because the anti-CD28 antibody of reticent type of the present invention is a kind of antibody, thereby it provides a kind of lasting effect, so that intermittently administration can obtain desired effect.As for the treatment phase,,, also can keep this tolerance even then stop using the anti-CD28 antibody of reticent type in case set up tolerance status.In this respect, other immunosuppressive drug that immunosuppressive action descended after the anti-CD28 antibody of this reticent type was better than stopping using undoubtedly.
Embodiment
Briefly described the present invention,, can further understand the present invention, provide described embodiment only in order to illustrate by with reference to some specific embodiment provided herein, and nonrestrictive, except as otherwise noted.Unless have a detailed description in addition,, implement the following examples with the well-known and conventional standard technique of those skilled in the art.
The determined amino acid sequence of embodiment 1 mouse anti human CD28 antibody
The hybridoma (clone: TN228, mouse IgG1 κ) that produces anti-people CD28 antibody is provided by doctor Yagita (Juntendo University School of Medicine, Japan)) generosity.Allow the anti-people CD28 antibody (TN228) of about 0.2mg purifying at the 0.64M Guanidinium hydrochloride, the 0.28M Tris-HCl of pH8.5,0.055M among the DTT, reach 90 minutes in 60 ℃ (in argon) reduction, carry out carboxymethylation by the interpolation iodoacetic acid to 0.13M in room temperature (in dark) and reach 45 minutes, succeeded by adding DTT to 0.32M (termination carboxymethylation reaction), and use a PD-10 post (catalog number (Cat.No.) 17-0851-01 immediately, Amersham Pharmacia Biotech, Uppsala Sweeden) makes it at the 0.1M sodium phosphate, carry out buffer-exchanged among the 0.002M EDTA of pH8.0.Eluate is transferred to 0.005MDTT, 0.02% glycerine; And 1/3rd (approximately 0.35ml) of this solution are transferred in the independent pipe, for the deblocking of heavy chain N-terminal.With 1800 μ U Pyrrolidonecarboxylic acid aminopeptidases (catalog number (Cat.No.) 7334, Takara Shuzo Co., Ltd., Tokyo, Japan) in 45 ℃ digestion these samples reach 24 hours.By 20 round-robin automatic edman degradations and with 241 type protein sequence determinators (Model 241 Protein Sequencer) (HewlettPackard, Palo Alto, CA) PTH analyzes, and measures the N-terminal sequence from the light chain and the heavy chain of deblocking sample.Analyze the PTH derivative with a Hypersil ODS C18 post.According to the specification sheets of manufacturers,, operate described sequenator and HPLC with the reagent, solvent and the post that derive from Hewlett Packard.
Result with the N-terminal order-checking of the TN228 of Pyrrolidonecarboxylic acid aminopeptidase deblocking is as follows:
| Residue number | Amino acid | Residue number | Amino acid | Residue number | Amino acid | Residue number | Amino acid |
| 1 | D，Q | 6 | Q，E | 11 | L | 16 | G，Q |
| 2 | l，v | 7 | s | 12 | A，V | 17 | Q，S |
| 3 | V，Q | 8 | P，G | 13 | V，A | 18 | R，L |
| 4 | L | 9 | A，P | 14 | S，P | 19 | A，S |
| 5 | T，K | 10 | S，G | 15 | L，S | 20 | T，I |
The clone of embodiment 2 variable region cDNA
Anchored PCR (PCR) method (Co with descriptions such as Co, M.S., N.M.Avadalovic, P.C.Caron, M.V.Avadalovic, D.A.Scheinberg and C.Queen.1992. have specific chimeric antibody and humanized antibody .J.Immunol.148:1149-1154 to CD33 antigen), from light chain and the heavy chain V district cDNA of described hybridoma cell clone TN228.Utilization is annealed to 3 ' primer in mouse κ chain C district and the γ chain C district respectively and is annealed to 5 ' primer on the G tail of cDNA of interpolation, increases on cDNA.For VLPCR, described 3 ' primer has following sequence (SEQ ID NO:14): 5 ' TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC 3 ' its residue 17-46 and mouse C
κDistrict's hybridization.For VH PCR, described 3 ' primer has following degenerate sequence (SEQ ID NO:15,16 and 17):
A G T
5′TATAGAGCTCAAGCTTCCAGTGGATAGACCGATGGGGCTGTCGTTTTGGC?3′
T
Its residue 17-50 and mouse IgG C
H1 hybridization.Non-hybridization sequences in two primer sets comprises the restriction enzyme site for clone's usefulness.With VL cDNA and VH cDNA subclone to TOPOII Blunt carrier (Invitrogen, Inc., Carlsbad, CA) in for sequencing.
From two polymerase chain reactions independently, several light chains and heavy chain clone are carried out sequencing.As for described light chain, identify two kinds and mouse variable region of light chain homologous unique sequences.A kind of VL sequence is accredited as unproductive allelotrope because phase shift mutation is non-functional.Another kind of VL sequence is a kind of typical case of functional mouse κ chain variable region.For described heavy chain, identify a kind of and typical murine heavy chain variable region homologous unique sequences.In Fig. 2 and Fig. 3, the nucleotide sequence of its variable region and the aminoacid sequence of inferring thereof have been described.
The construction and expression of embodiment 3 chimeric TN228-IgG2M3
(method)
According to (He such as He, X.Y., Z.Xu, J.Melrose, A.Mullowney, M.Vasquez, C.Queen, V.Vexler, C.Klingbeil, M.S.Co and E.L.Berg.1998. select albumen and P-to select albumen that the humanization and the pharmacokinetics .J.Immunol.160:1029-1035 of specific monoclonal antibody are arranged to E-) described, by PCR, with the V of TN228
LAnd V
HBe transformed into little exon section in abutting connection with the XbaI site; And with its subclone in described light chain and heavy chain expression plasmid (Fig. 1).Each little exon comprises a segment signal peptide sequence, one section ripe variable region sequences and one section donor splicing site sequence that derives from the highest mouse J chain gene of homology.With such donor splicing site sequence, with the exon montage of described V district on the constant region of people's antibody.After being cloned into little exon in the expression vector, measure the sequence of each little exon, obtain correct sequence and do not produce the PCR mistake guaranteeing.Equally, by sequencing, confirm the described constant region exon of light chain and heavy chain expression plasmid.
In this manual, ChTN228 is meant a kind of mouse TN228VL and VH variable region, the CH of human IgG2 M3 and chimeric antibody of people's light chain κ constant region of comprising.From ethnic group is that this CH of 2 genomic fragments is modified (Cole, M.S., human IgG2's varient of C.Anasetti and J.Y.Tso.1997. inosculating antibody CD33 does not have mitogenesis .J.Immunol.159:3613-3621 to the T cell), and this light chain to derive from ethnic group be the K genomic fragment.Described heavy chain gene and light chain gene are all driven by the main immediate early promoter and the enhanser of human cytomegalic inclusion disease virus.Fetch the transcription terminator (Ashfield that comes from people's complement gene C 2 behind the described heavy chain gene, R., the Transcription Termination between the closely linked people's complement of P.Enriquez-Harris and the N.J.Proudfoot.1991. gene C 2 and the B factor: the common terminator factor of C2 and c-myc? EMBO J.10:4197-4207).Light chain selective marker gpt gene (Mulligan, R.C. express the selection .Proc.Natl.Acad.Sci.USA78:2072-2076 of the zooblast of colibacillary xanthine-guanine phosphoribosyl transferase encoding gene with P.Berg.1981.) and heavy chain selective marker dhfr gene (Simonsen, the mouse Tetrahydrofolate dehydrogenase cDNA of a C.C. and A.D.Levinson.1983. reconstruction separates and expresses .Proc.Natl.Acad.Sci.USA 80:2495-2499) all drive by the SV40 early promoter.In order to express chimeric TN228, finish the transient transfection of transfection in the COS-7 cell (monkey-kidney cells system) with lipofection amine (catalog number (Cat.No.) 10964-013, GIBCO BRL).With the specific antibody of the anti-human IgG γ of goat chain as capture agent, and with the anti-people κ of the goat of puting together HRP chain antibody as colouring reagents, by ELISA, to the sub-exhausted substratum of transient transfection analyst IgG2M3 production of antibodies.In addition, also by indirect IF staining, described exhausted substratum test ChTN228 is attached to P815/CD28
+Ability on the cell (with the stable transfected cells of CD28 transfection P815 (mouse hypertrophy cell knurl) gained); And analyze with flow cytometer.As for the generation of stable cell lines, then by electroporation, making the chimeric expression plasmid transfection is among the Sp2/0 to rat bone marrow tumour cell; And transfectant is picked out in the expression according to gpt.About transient transfection,, analyze the sub-exhausted substratum of stable transfection by ELISA.
The result
By PCR, with clone's V
LGene and V
HGenetic modification becomes little exon (Fig. 2 and Fig. 3), and with its subclone in light chain above-described and shown in Figure 1 and heavy chain expression carrier.
The transient transfection of COS-7 cell: making described chimeric expression carrier transient transfection is among the COS-7 to monkey-kidney cells, to produce chimeric TN228
+Antibody.And check the chimeric IgG2M3 production of antibodies situation of described transfectional cell exhausted substratum by ELISA; And P815/CD28
+Check described transfectional cell exhausted substratum and P815/CD28 with flow cytometer
+Cell bonded situation.In two kinds of analyses, the exhausted substratum all is a male.Output from the chimeric antibody of transient transfection approximately is 0.9 μ g/ml.ChTN228 antibody from instantaneous supernatant liquor is attached to P815/CD28 in the mode that relies on concentration
+On the cell (data not shown).
The stable transfection of Sp2/0 cell: in order to produce stable clone, and with the transfection of described chimeric expression carrier in the Sp2/0 cell.Check the situation that the TN228 chimeric antibody produces in several transfectant exhausted substratum; And as described transient transfection, check and P815/CD28
+Cell bonded situation.For two kinds of analyses, most of transfectants are male.The antibody producing rate is higher is selected owing to it for a kind of transfectant, and it is cultivated enlarges and it is grown in 5 liters serum free medium.By affinity chromatography, from 5 liters of exhausted substratum, be purified into ChTN228.Antibody purified output approximately is 25mg.
The protein purification of embodiment 4 chimeric antibody ChTN228
5 liters GIBCO hybridoma serum free mediums (catalog number (Cat.No.) 12045-076, GIBCOBRL) in, cultivate ChTN228 from stable transfection and express a kind of (clone 7H) in the high transfectant.When cell survival reaches 10% or when lower, results exhausted culture supernatant; And it is concentrated into 500ml, and with a Pharmacia P1 pump (2-3ml/ minute), on the albumen-A Sepharose post with its application of sample to one 5ml.Wash this post with PBS, use 0.1M glycine, the described antibody of 0.1M NaCl pH2.7 wash-out then.To the albumen of 2 liters of PBS dialysis institute wash-outs, and replacing PBS reaches 3 times; Then, it is added to one with on the PBS equilibrated PD-10 post that contains extra 0.1M NaCl, makes its desalination.Being stored in before 4 ℃,, filter and take off the protein solution of supersalt by the filter paper of a 0.2mm.
Use the Perkin Elmer HPLC system that forms by a PE ISS 200 Advanced LC Sample Processor (sample processor), PE Series 410 Bio LC Pump (pump), a PE 235C Diode ArrayDetector (diode-array detector) and PE Nelson 600 Series LINK, carry out size exclusion HPLC.Use Perkin ElmerTurbochrom Navigator Version 4.1 softwares, control automatic sampler, pump and detector; And with this software obtain, storage and processing data.With two TosoHaasTSK-GEL G3000SWXL size exclusion HPLC posts that are connected in series, finish separation; The data of described post are: 7.8mm * 300mm, and the granularity of 5mm, the pore size of 250 (catalog number (Cat.No.) 08541, TosoHaas, Montgomeryville, MD).Moving phase is 200mM potassiumphosphate/150mM Repone K of pH6.9, and flow velocity is 1.00ml/ minute.Under 220nm wavelength and 280nm wavelength, monitor the elutriant of this post with spectrophotometer.The volume injected of ChTN228 sample is 50 μ l (50 μ g).
According to standard step, (SanDiego CA) carries out SDS-PAGE to the gradient gel of usefulness 4-20% for catalog number (Cat.No.) EC6025, Novex.
By size exclusion HPLC and SDS-PAGE, analyze the purity of isolating ChTN228.According to this analysis, this albumen is 96.5% monomer, and has the proteic mobility of the about 160kD of the molecular weight of being equivalent to.MuTN228, isotype contrast MuFd79 (mouse IgG1), ChTN228 and the SDS-PAGE of isotype contrast HuEP5C7 (human IgG2 M3) under non-reduced condition analyze and also show: all four kinds of antibody all have the molecular weight of about 150-160kD.Analyzing four kinds of same albumen under reductive condition then shows: all four kinds of antibody all are made up of the light chain of the heavy chain of an about 50kD of molecular weight and an about 25kD of molecular weight.
Embodiment 6 competitive assay
Method
Use MuTN228-FITC antibody from 250ng/ two times of serial dilutions of on-test, finish titration experiments.On ice with P815/CD28
+Cell (5 * 10
5Individual cell/test) hatches with the antibody of FITC mark and reach 1 hour, then with the PBS washing and use flow cytometry analysis.As for competitive assay, 25ng MuTN228-FITC and competitive ChTN228 or MuTN228 antibody from 800ng/ two times of serial dilutions of on-test, are added to P815/CD28
+Cell (5 * 10
5Individual cell/test) in.In contrast, MuTN228-FITC and P815/CD28 that 25ng is independent
+Cell (5 * 10
5Individual cell/test) hatches (promptly without any competitor) together.Equally, test is as the HuEP5C7 isotype control antibodies (800ng/ test) and the MuFd79 isotype control antibodies (800ng/ test) of competitor.On ice (in dark), cell hatched with the final volume of 150ml with mixtures of antibodies reach 1 hour, washing then, and use flow cytometry analysis.
The result
Described in method, use the flow cytometry competitive assay, relatively the binding specificity of MuTN228 antibody and ChTN228 antibody.Difference is measured unlabelled MuTN228 or ChTN228 mixes mutually with the MuTN228 antibody of 25ng FITC mark, and make itself and P815/CD28 ' cell incubation.MuTN228 and ChTN228 be mode and the MuTN228-FITC competition to rely on concentration, and this shows that antigenic combination of these two kinds of antibody and CD28 is specific (Fig. 4).Isotype control antibodies MuFd79 and HuEP5C7 with MuTN228-FITC competition, show that MuTN228 antibody and ChTN228 antibody discerns CD28 antigen by the specificity interaction in V district.
The inosculating antibody people CD28 antibody that 7 couples of people FR of embodiment affinity reduces suppresses first to be mixed
Close lymphocyte reaction
The preparation of cell
By (Amersham Pharmacia Biotech, Tokyo Japan) carries out density gradient centrifugation, prepares the human peripheral blood mononuclear cell (PBMC) from the normal volunteer of health with Ficoll-Paque plus.With isopyknic RPMI1640 dilution human blood, be layered on then Ficoll-Paque plus above.In room temperature after centrifugal 30 minutes, collect the peripheral blood cells monocyte and wash with RPMI1640.After this, with substratum (containing 2.5% people AB type serum, 2 mercapto ethanol and antibiotic RPMI1640) suspension peripheral blood cells monocyte, and with its be added to a nylon fiber post (Wako junyaku, Osaka, Japan) on.At 5%CO
2In in 37 ℃ hatch 1 hour after, with warm substratum wash-out T cell.
In mixed lymphocyte reacion, human B cell system (Raji and JY) is used as the yield stimulant cell.These cells are process x-ray bombardment (2000R) before using.
First mixed lymphocyte reacion (first MLR)
The human T-cell (1 * 10 of purifying
5Individual cells/well) and the Raji (1 * 10 that shone
5Individual cells/well) is inoculated in the flat small plate in 96 holes.In substratum, add microbiotic, and culturing cell 7 days.In last 6 hours cultivate, usefulness 10kBq/ hole [
3H] all cultures of thymidine (AmershamPharmacia biotech) mark.Harvested cell, and the radioactivity of mixing with liquid scintillation counter measurement.
In Fig. 5 and Fig. 6, shown the influence of TN228-IgG2m3 (ChTN228) to first MLR.The anti-people CD28 of primary antibody TN228 (MuTN228) does not suppress first MLR, yet chimeric antibody TN228-IgG2m3 suppresses first MLR in the mode of dependent dose.Therefore, the Fc district of anti-people CD28 antibody is transformed into the Fc district that people Fc R affinity is reduced, then makes this antibody become that cell proliferation has antagonism to T.
The anti-people CD28 chimeric antibody of people Fc R affinity reduction has been weakened the low responsiveness of T cell in the secondary mixed lymphocyte reacion.
Secondary mixed lymphocyte reacion (secondary MLR)
The human T-cell (1 * 10 of purifying
5Individual cells/well) and the Raji cell (1 * 10 that shone
5Individual cells/well) is inoculated in the flat small plate in 96 holes.In substratum, add microbiotic and incubated cell.After 5 days, collecting cell is with fresh substratum washing.With fresh substratum suspension cell, and cultivated described cell 8 days.Stimulate described cell again with Raji cell that shone or JY cell.After cultivating again 7 days, with the 10kBq/ hole [
3H] thymidine hatched 6 hours with cell.Harvested cell, and use the liquid scintillation counter measurement radioactivity.
TN228-IgG2m3 suppresses first MLR (Fig. 5 and Fig. 6).Then, we analyze the influence of this antibody to secondary MLR.Described antibody is added in the first MLR culture, then, from culture supernatant, removes this antibody.In not having the substratum of antibody after the culturing cell, with same irritation cell (Raji) or third party's stimulator (JY) irritation cell again.Compare with the propagation of the cell of not handling, the propagation of the cell of handling by first MLR with TN228-IgG2m3 reduces.But with third party's stimulator (JY), two kinds of cell proliferations are to almost same degree (Fig. 7).This result has shown that the anti-people CD28 antibody that people Fc R affinity is reduced can pass through allos (alo-) antigenic stimulation and the vigor (energy) of inducing T cell.
The design of embodiment 8 humanization TN228 variable regions
By microcomputer modelling, analyze the V region sequence of MuTN228.Based on to Kabat antibody sequence database (8.Johnson, G. with T.T.Wu.2000.Kabat database and application thereof: sequence homology search 30 years .Nucleic Acids Res.28:214-218 behind first amplitude variation opposite sex figure), pick out IC4 (Manheimmer-Lory, A., J.B.Katz, M.Pillinger, C.Ghossein, A.Smith, B.Diamond.1991. the characterization of molecules .J.Exp.Med.174:1639-1652 that has the antibody of the relevant idiotype of anti-DNA), coming provides framework for variable region of heavy chain and the variable region of light chain of humanization TN228.Humanized TN228 variable region of heavy chain has 65 residues identical with the residue of mouse TN228 heavy chain framework in 85 framework residues, and 76% sequence identity is promptly arranged.Humanized TN228 variable region of light chain has 56 residues identical with the residue of mouse TN228 light chain framework in 80 framework residues, and 70% sequence identity is promptly arranged.
The program that uses a computer ABMOD and ENCAD (Levitt, M.1983. the computer simulation .J.Mol.Biol.168:595-620 of the molecular dynamics .I. track of native protein) make up the molecular model of TN228 variable region; Thereby with this model determine in the mouse TN228 framework close enough CDR may with they interactional amino acid whose positions.For variable region of heavy chain and the variable region of light chain that designs humanization TN228, will be transplanted to from the CDR of mouse TN228 heavy chain in the people IC4 heavy chain framework region, and the CDR from mouse TN228 light chain will be transplanted in the framework region of people IC4 light chain.In the framework position that the noticeable and described CDR of computer model prompting contacts, from the aminoacid replacement of mouse antibodies the amino acid of primitive man's framework.For humanized TN228, finish this replacement at heavy chain residue 27,29,30,48,67,71 and 78 parts.For light chain, do not replace (being about to MuTN228CDR is grafted directly in the IC4 framework region).In addition, as for being rare framework residue on the corresponding position in people's antibody database, then replace with the locational people's consensus sequence of those residues amino acid.For humanized TN228,, carry out this replacement in heavy chain residue 23,40,73,83 and 85 parts and in light chain residue 69 and 77 parts.Fig. 9 and Figure 10 have shown the variable region of heavy chain of humanization TN228 antibody and the aminoacid sequence of variable region of light chain.
Embodiment 9 humanization TN228-IgG2M3 construction and expression
Method
In case designed described humanization amino acid sequences as mentioned above, then make up gene and encode them, comprise signal peptide, donor splicing site signal and suitable restriction enzyme sites (Fig. 8).Make up heavy chain variable region gene and chain variable region gene, and be increase their (He of 8 kinds of overlapping synthetic oligonucleotides of about 65-80 base with length, X.Y., Z.Xu, J.Melrose, A.Mullowney, M.Vasquez, C.Queen, V.Vexler, C.Klingbeil, M.S.Co and E.L.Berg.1998. select albumen and P-to select albumen that the humanization and the pharmacokinetics .J.Immunol.160:1029-1035 of specific monoclonal antibody are arranged to E-).Described oligonucleotide is annealed in pairs, and it is extended, produce four kinds of double-stranded fragments with the Klenow fragment of dna polymerase i.With the fragment sex change that is produced, anneal in pairs, and it is extended with the Klenow fragment, produce two kinds of fragments.These fragment sex change, annealing in pairs, and again it is extended, and the gene of generation total length.The product that is produced will increase with the Taq polysaccharase by polymerase chain reaction (PCR), use gel-purified, and digest with XbaI; Use gel-purified once more, subclone is to the XbaI site that is used for expressing the pVg2M3 of heavy chain and being used to express the pVk of light chain then.The previous pVg2M3 carrier (Cole that is used for people γ 2 heavy chain expressions that described, M.S., human IgG2's varient of C.Anasetti and J.Y.Tso.1997. inosculating antibody CD33 does not have mitogenesis .J.Immunol.159:3613-3621 to the T cell) and be used for the pVk carrier (Co that human kappa light chain is expressed, M.S., N.M.Avadalovic, P.C.Caron, M.V.Avadalovic, D.A.Scheinberg and C.Queen.1992. have specific chimeric antibody and humanized antibody .J.Immunol.148:1149-1154 to CD33 antigen).
By nucleotide sequencing, verify the V district of final heavy chain plasmid and light chain plasmid and the sequence of constant region exon.By restriction endonuclease mapping, verify the entire structure of final plasmid.Carry out all operations of DNA by standard method.
In this manual, HuTN228 is meant and comprises humanization TN228V
HAnd V
LThe humanized antibody of the CH of variable region, human IgG2 M3 and people's light chain κ constant region.From ethnic group is that this CH of 2 genomic fragments is modified (Cole, M.S., human IgG2's varient of C.Anasetti and J.Y.Tso.1997. inosculating antibody CD33 does not have mitogenesis .J.Immunol.159:3613-3621 to the T cell), and this light chain to derive from ethnic group be the K genomic fragment.The main immediate early promoter of human cytomegalic inclusion disease virus and enhanser had not only driven heavy chain gene, but also had driven light chain gene.Described heavy chain gene back is the transcription terminator (Ashfield that derives from people's complement gene C 2, R., the Transcription Termination between the closely linked people's complement of P.Enriquez-Harris and the N.J.Proudfoot.1991. gene C 2 and the B factor: the common terminator factor of C2 and c-myc? EMBO J.10:4197-4207).Light chain selective marker gpt gene (Mulligan, R.C. express the selection .Proc.Natl.Acad.Sci.USA78:2072-2076 of the zooblast of colibacillary xanthine-guanine phosphoribosyl transferase encoding gene with P.Berg.1981.) and heavy chain selective marker dhfr gene (Simonsen, the mouse Tetrahydrofolate dehydrogenase cDNA of a C.C. and A.D.Levinson.1983. reconstruction separates and expresses .Proc.Natl.Acad.Sci.USA 80:2495-2499) both drive by the SV40 early promoter.
In order to express HuTN228, (catalog number (Cat.No.) 11668-027 LifeTechnologies) is accomplished to transient transfection in the COS-7 cell (monkey-kidney cells system) with lipofection amine 2000.With the specific antibody of the anti-human IgG γ of goat chain as capture agent, and with the anti-people κ of the goat of puting together HRP chain antibody as colouring reagents, by ELISA, come the sub-exhausted substratum of transient transfection is carried out the analysis of human IgG2 M3 antibody generation aspect.In addition by indirect IF staining, to described exhausted substratum check HuTN228 and P815/CD28
+Cell bonded ability; And use flow cytometer, described exhausted substratum is analyzed (data not shown).As for the generation of stable cell lines, making humanized expression plasmid transfection by electroporation is among the Sp2/0 to rat bone marrow tumour cell; And transfectant is selected in the expression according to gpt.Aspect transient transfection,, analyze the sub-exhausted substratum of stable transfection by ELISA.
The result
Based on the design of humanization V region amino acid sequence, described in method, make up the V district gene of heavy chain and the V district gene (Fig. 9 and Figure 10) of light chain.Just as shown in Figure 8, the V district gene of heavy chain and the V district gene of light chain are cloned into respectively in pVg2M3 carrier and the pVk carrier.Analyze several clones by nucleotide sequencing, and the correct clone of heavy chain expression carrier and light chain expression vector is used for transfection.Equally, by sequencing, further confirm the constant region of heavy chain expression carrier and light chain expression vector.
The expression of embodiment 10 HuTN228
The transient transfection of COS-7 cell: making described expression vector transient transfection is among the COS-7 to monkey-kidney cells, to produce HuTN228 antibody.With institute's transfectional cell exhausted substratum, come by ELISA check humanization IgG2M3 production of antibodies, and check and P815/CD28 with flow cytometer
+The combination of cell (data not shown).In two kinds of analyses, the exhausted substratum is a male.The output of the humanized antibody that is come by transient transfection approximately is 3.7g/ml.HuTN228 antibody from instantaneous supernatant liquor is attached to P815/CD28 in the mode that relies on concentration
+On the cell (data not shown).
The stable transfection of Sp2/0 cell: in order to produce stable clone, with the transfection of described humanization expression vector in the Sp2/0 cell.As described transient transfection, to several parts of substratum checks of several transfectant exhausted HuTN228 production of antibodies.A kind of transfectant (clone 4) is selected owing to the antibody producing rate is higher, and in GIBCO hybridoma serum free medium with its enlarged culturing.By affinity chromatography, from 570 milliliters of exhausted substratum, be purified into HuTN228 antibody.Antibody purified output approximately is 7mg.
Embodiment 11 proteic purifying
570ml GIBCO hybridoma serum free medium (catalog number (Cat.No.) 12045076, LifeTechnologies) in, cultivate from stable transfection (clone 4), HuTN228 expresses a kind of in the high transfectant.When cell survival reaches 10% or when lower, results exhausted culture supernatant; And on the albumen-A Sepharose post with its application of sample to one 2ml.Wash this post with PBS, use 0.1M glycine, the described antibody of 0.1M NaCl pH2.5 wash-out then.To the albumen of 2 liters of PBS dialysis institute wash-outs, and replacing PBS reaches 3 times; Then, one with the PBS equilibrated PD-10 post that contains extra 0.1M NaCl on, make its desalination.Being stored in before 4 ℃,, filter and take off the protein solution of supersalt by the filter paper of a 0.2mm.
Embodiment 11 carries out purity testing by size exclusion HPLC and SDS-PAGE
Method
Use the Perkin Elmer HPLC system that forms by a PE ISS 200Advanced LC Sample Processor (sample processor), a PE Series 410Bio LC Pump (pump), a PE 235C Diode ArrayDetector (diode-array detector) and PE Nelson 600 Series LINK, carry out size exclusion HPLC.Use Perkin ElmerTurbochrom Navigator Version 4.1 softwares, control automatic sampler, pump and detector; And with this software obtain, storage and processing data.With two TosoHaasTSK-GEL G3000SWXL size exclusion HPLC posts that are connected in series (7.8mm * 300mm, the granularity of 5mm, the pore size of 250 , catalog number (Cat.No.) 08541, TosoHaas, Montgomeryville MD), finishes separation.Moving phase is 200mM potassiumphosphate/150mM Repone K of pH6.9, and flow velocity is 1.00ml/ minute.Under 220nm wavelength and 280nm wavelength, monitor the elutriant of this post with spectrophotometer.The volume injected of HuTN228 sample is 60l (60g).
According to standard step, (SanDiego carries out SDS-PAGE on CA) for catalog number (Cat.No.) EC6025, Novex at the gradient gel of 4-20%.
According to manufacturer's recommendation, with Human IgG Subclass Profile ELISA Kit (human IgG subclass profile ELISA test kit) (catalog number (Cat.No.) 99-1000, Zymed Laboratories, South San Francisco, CA), confirm the isotype of purified antibody.
The result
By size exclusion HPLC and SDS-PAGE, analyze the purity of isolating HuTN228 antibody.The HPLC elution profile that does not show HuTN228.According to this analysis, this albumen is about 98% monomer, and has the proteic mobility of the about 160kD of the molecular weight of being equivalent to.
MuTN228, isotype contrast MuFd79 (mouse IgG1), HuTN228 and the SDS-PAGE of isotype contrast HuEP5C7 (human IgG2 M3) under non-reduced condition analyze and also show: all four kinds of antibody all have the molecular weight of about 150-160kD.Analyzing four kinds of same albumen under reductive condition then shows: all four kinds of antibody all are made up of the light chain of the heavy chain of an about 50kD of molecular weight and an about 25kD of molecular weight.
The isotype test shows: the isotype of HuTN228 antibody and desired IgG2 isotype consistent (data not shown).
Embodiment 12 FACS competitive assay
Method
Use MuTN228-FITC antibody from 250ng/ two times of serial dilutions of on-test, finish titration experiments.On ice at 1001FACS dyeing damping fluid (FSB=PBS, 2%FBS, 3% normal mouse serum, 0.1%NaN
3) in, with the antibody and the P815/CD28 of FITC mark
+Cell (3 * 10
5Individual cell/test) hatch together and reach 1 hour, then with the FBS washing of 2ml, and with flow cytometry analysis (data not shown).
As for competitive assay, (from the antibody of 200g/ml) of three times of serial dilutions of competitive HuTN228 antibody among the MuTN228-FITC among the 251FSB (50ng/ test) and, the 25lFSB or MuTN228 antibody is mixed, and add P815/CD28 in the 50lFSB to
+Cell (3 * 10
5Individual cell/test) in.In contrast, hatch P815/CD28 with MuTN228-FITC separately
+Cell (the 50ng/ test is in the FSB of 50l).Equally, test is as HuEP5C7 (human IgG2 M3) isotype control antibodies non-specific competitor, in 25l FSB (200g/ml) and MuFd79 (mouse IgG1) isotype control antibodies (200g/ml).On ice (in dark), in final volume 100l, mixtures of antibodies hatched with cell and reach 1 hour, then with 2ml FSB washing, and use flow cytometry analysis.Repeat this experiment three times.
The result
As described in method,, MuTN228 antibody and HuTN228 antibody and P815/CD28 have been compared with a flow cytometry competitive assay
+The binding specificity of the CD28 molecule on the cell.In Fig. 5, shown representational result.MuTN228 and HuTN228 both mode and MuTN228-FITC competitions to rely on concentration, this shows that these two kinds of antibody are specific with antigenic combination of CD28.The relative combination of HuTN228 is the relative bonded part of MuTN228.Isotype control antibodies MuFd79 and HuEP5C7 with MuTN228-FITC competition, show that MuTN228 antibody and HuTN228 antibody discerns CD28 antigen by the specificity interaction in V district.
Embodiment 13 ELISA competitive assay
Method
With the sCD28-Fc in 100l/ hole (in PBS, 0.5g/ml) (sCD28-Fc is that the extracellular domain of wherein CD28 and CH2 structural domain and the CH3 structural domain bonded of IgG1 refer to fusion rotein) at 4 ℃ of bags by 96 hole elisa plate (Nunc-Immuno plates, catalog number (Cat.No.) 439454, NalgeNunc, Naperville IL) spends the night.With the SuperblockBlocking Buffer 300l/ hole, in TBS (sealing damping fluid) (catalog number (Cat.No.) 37535, Pierce, Rockford, IL) seal this flat board and reach 30 minutes, and with the ELISA ELISA Wash Buffer (lavation buffer solution) in 300l/ hole (EWB=PBS 0.1%Tween-20) washs described flat board.By final volume 200l/ hole, three parts every kind ground add 100l ELISA Buffer (ELISA damping fluid) (EB=PBS, 1%BSA, 0.1%Tween-20) mixture of the MuTN228-vitamin H (0.5g/ml) in and competitor HuTN228 antibody or the three times serial dilutions (from 100g/ml) of MuTN228 antibody in 100lEB.Test equally in 100lEB, as the isotype control antibodies HuEP5C7 and the MuFd79 (100g/ml) of non-specific competitor.The EB of 100l is added in the MuTN228-vitamin H (0.5g/ml) of 100l, contrasts as ' uncontested dose '.The EB of 200l is added in the remaining hole (do not comprise the MuTN228-vitamin H), as blank (contrast).Reach 1.5 hours at this flat board of room temperature vibration insulation.After the EWB with the 300l/ hole washs described hole 4 times, in all holes, add the 100l/ hole Streptavidin-HRP (1g/ml, catalog number (Cat.No.) 21124, Pierce).Reach 1 hour at this flat board of room temperature vibration insulation.Wash as mentioned above after the described hole, and the ABTS substrate in interpolation 100l/ hole in all holes (catalog number (Cat.No.) 507602 and 506502, KPL, Gaithersburg, MD).At room temperature this flat board of incubation reaches 5 to 7 minutes, the optical density(OD) of reading 415nm then.Repeat this experiment three times.
The result
As described in method,, compared the binding specificity of HuTN228 antibody and MuTN228 antibody and sCD28-Fc with a kind of ELISA competitive assay.In Figure 12, shown representational result.MuTN228 and HuTN228 both mode and competitions of MuTN228-vitamin H to rely on concentration.Isotype control antibodies MuFd79 and HuEP5C7 with MuTN228-vitamin H competition, show that MuTN228 antibody and HuTN228 antibody discerns CD28 antigen by the specificity interaction in V district.The MuTN228 of all three times experiments and the IC of HuTN228 have been displayed in Table 2
50Value.The relative combination of HuTN228 on average is a MuTN228 bonded 1/2.6th.
The summary of table 2 ELISA competition
IC
50(g/ml)
| | Experiment | 1 | | | Mean value | Standard deviation |
| MuTN228 | 0.21 | 0.20 | 0.15 | 0.19 | 0.03 | |
| HuTN228 | 0.37 | 0.64 | 0.48 | 0.50 | 0.14 |
Method
Method (Queen according to Queen etc., C., W.P.Schneider, H.E.Selick, P.W.Payne, N.F.Landolfi, J.F.Duncan, N.M.Avdalovic, M.Levitt, R.P.Junghans, the humanized antibody .Proc.Natl.Acad.Sci.86:10029-10033 of one kind of T.A.Waldmann.1989. and interleukin-22 receptors bind), measured the relative binding affinity of MuTN228 antibody and HuTN228 antibody.Speak briefly, make 50l binding buffer liquid (Binding Buffer) (BB=PBS, 2%FBS, 1g/ml mouse IgG, 0.1%NaN
3) in about 10ng
125Three times of serial dilutions (from 400g/ml) in 501BB mix the MuTN228 of I mark mutually with competitor MuTN228 antibody or HuTN228 antibody, and every kind of mixing is done three parts; Then, it is added to incubation tube (Skatron Macrowell Tube Strips, catalog number (Cat.No.) 15773, Molecular Devices, Sunnyvale, CA) the 100l P815/CD28 in
+Cell (2.5 * 10
5Individual cell/test) in; And hatch in 4 ℃ of gentle vibrations and to reach 90 minutes.Test equally in 50lBB, as the isotype control antibodies HuEP5C7 and the MuFd79 (400g/ml) of non-specific competitor.After hatching, cell-mixtures of antibodies is transferred to centrifuge tube (the Sarstedt MicroTubes that contains 0.1ml 80% dibutyl phthalate-20% sweet oil, catalog number (Cat.No.) 72.702, Sarstedt, Newton, NC) in, BB with 50l washs described incubation tube once, and by centrifugal counting and free counting (Kuziel, the W.A. that comes separation and combination of the method for having described, S.J.Morgan, T.C.Dawson, S.Griffin, O.Smithies, the serious minimizing .Proc.Natl.Acad.Sci.94:12053-12058 that K.Ley, N.Maeda.1997. leukocyte adhesion and monocyte in the mouse that CC-chemokine receptor 2 lacks exosmose).Repeat this experiment three times.
The result
As described in the method, use
125I traget antibody competitive assay has compared the relative binding affinity of MuTN228 antibody and HuTN228 antibody.In Figure 13, shown representational result.MuTN228 and HuTN228 both with the mode that relies on concentration with
125The MuTN228 competition of I mark.Isotype control antibodies MuFd79 when high density, demonstrate weak and repeatably the competition, but isotype control antibodies HuEP5C7 not with
125The MuTN228 competition of I mark; This shows that the specificity of HuTN228 antibody by the V district interacts and discerns CD28 antigen.The MuTN228 of all three times experiments and the IC of HuTN228 have been displayed in Table 3
50Value.The apparent binding affinity of HuTN228 is about 2.4 times of apparent binding affinity of MuTN228 antibody.
Table 3
125The summary of I competition
IC
50(nM)
| | Experiment | 1 | | | On average | Standard deviation |
| MuTN228 | 0.93 | 1.05 | 1.02 | 1.00 | 0.06 | |
| HuTN228 | 2.65 | 2.43 | 2.13 | 2.40 | 0.26 |
The determined amino acid sequence of the anti-mouse CD28 of embodiment 15 hamsters antibody
Method
Hybridoma and antibody: the hybridoma PV1 that obtains the anti-mouse CD28 of armenian hamster from ATCC (ATCC HB-12352).From Southern Biotechnology (Birmingham, AL) buy purifying PV1, put together the PV1 of R-phycoerythrin (R-PE).The anti-CD28 antibody 37.51 of Syria hamster from PharMingen (San Diego, CA).Second antibody put together fluorescein (FITC) the anti-armenian hamster IgG of donkey (H+L), put together FITC the anti-Syria hamster IgG of donkey (H+L), put together the anti-mouse IgG of donkey (H+L), the R-PE-F (ab ') of FITC
2The anti-mouse IgG of donkey (H+L), then from Jackson ImmunoResearch (West Grove, PA); Put together the goat anti-mouse κ of FITC, the goat anti-mouse κ that puts together the goat anti-mouse IgG 3 of R-PE and put together horseradish peroxidase (HRP) then from Southern Biotechnology.Goat anti-mouse IgG 3 and mouse IgG3 isotype contrast FLOPC 22, then from SigmaChemicals (St.Louis, MO).In our laboratory, prepare the anti-mouse CD3 of armenian hamster antibody 145.2C11 and hamster thereof/mouse mosaic 145.2C11-IgG3.The 145.2C11 that puts together FITC from Boehringer Mannheim (Indianapolis, IN).
The clone of variable region cDNA: with anchored PCR (PCR) method (Co of descriptions such as Co, M.S., N.M.Avadalovic, P.C.Caron, M.V.Avadalovic, D.A.Scheinberg and C.Queen.1992.J.Immunol.148:1149-1154), from the light chain of described hybridoma cell clone PV1 and the cDNA in heavy chain V district.Utilization is annealed to 3 ' primer in hamster κ chain C district and the γ chain C district respectively and is annealed to 5 ' primer on the G tail of cDNA of interpolation, increases on cDNA.For V
LPCR, described 3 ' primer have sequence 5 ' TATAGAGCTCCACTTCCAGTGCCC 3 ' (SEQ ID NO:21), its residue 11-24 and hamster C
κDistrict's hybridization.For V
HPCR, described 3 ' primer have following degenerate sequence (SEQ ID NO:18,19 and 20):
A G T
5′TATAGAGCTCAAGCTTCCAGTGGATAGACCGATGGGGCTGTCGTTTTGGC，
T
The IgG C of its residue 19-50 and most of rodents
H1 hybridization.Non-hybridization sequences in two kinds of primer sets comprises the restriction site for clone's usefulness.The cDNA subclone of VL and VH is supplied sequencing in the pUC19 carrier.For fear of the mistake that PCR produces, every kind of cDNA has measured the sequence of 5 independent clonings; And only select clone's the sequence clone consistent, express chimeric PV1 with consensus sequence.
The result
The clone of PV1V district cDNA:, from hybridoma, cloned the cDNA of PV1 variable region of light chain and the cDNA of variable region of heavy chain as described in method.For VLPCR, only corresponding to hamster C
γ3 ' the primer in district can produce V from PV1
LThe cDNA product.On the other hand, from hamster C
γA kind of 3 ' primer in district does not produce the product of any PCR.These results show: the light chain of hybridoma PV1 uses κ.Measured several clones' of light chain and heavy chain sequence, found that they contain identical V respectively
LAnd V
HC
H1 and C
γThe finite sequence data show: heavy chain of being cloned and light chain do not originate from mouse.
The construction and expression of embodiment 16 chimeric PV1-IgG3
Method
According to the method for having described (He, X.Y., Z.Xu, J.Melrose, A.Mullowney, M.Vasquez, C.Queen, V.Vexler, C.Klingbeil, M.S.Co and E.L.Berg.1998.J.Immunol.160:1029-1035), by PCR, with the V of PV1
LAnd V
HBe prepared into little exon section in abutting connection with the XbaI site; And they are incorporated into respectively in light chain expression plasmid and the heavy chain expression plasmid (Figure 14).Each little exon comprises a segment signal peptide sequence, one section ripe variable region sequences and one section 5 ' donor splicing site sequence that derives from the highest mouse J chain gene of homology.With such donor splicing site, with the exon montage of described variable region on the constant region of mouse antibodies.After being cloned into little exon in the expression vector, measuring the sequence of each little exon again, thereby guarantee to introduce correct splicing signal, and do not produce the mistake of PCR.
Make up a kind of carrier, a kind of single plasmid of cause is expressed heavy chain gene and the light chain gene of chimeric PV1-IgG3.In this report, PV1-IgG3 represents a kind of hamster PV1VL and V of comprising
HThe chimeric antibody of the κ constant region of the CH of variable region, mouse IgG3 and mouse light chain.By being similar to two step cloning of described methods (Cole, M.S., C.Anasetti and J.Y.Tso.1997.J.Immunol.159:3613-3621) such as Cole, obtain expression vector pV1.g3.rg.dE (Figure 14).CH derives from mouse γ 3 genomic fragments, and light chain is from the κ fragment.Described heavy chain gene and light chain gene are all driven by the main immediate early promoter and the enhanser of human cytomegalic inclusion disease virus, and they are derived from the transcription terminator (Ashfield of people's complement gene C 2, R., P.Enriquez-Harris and N.J.Proudfoot.1991.EMBO are J.10:4197-4207) separate.The gpt gene of selective marker (Mulligan, R.C. and P.Berg.1981.Proc.Natl.Acad.Sci.USA 78:2072-2076) is driven by modified SV40 early promoter.In order to express chimeric PV1-IgG3, be among the NS0 to rat bone marrow tumour cell with the transfection of described single plasmid carrier; And transfectant is selected in the expression according to gpt.With goat anti-mouse IgG 3 as capture agent, and with the goat anti-mouse κ chain of puting together HRP as colouring reagents, by ELISA, transfectant exhausted substratum is carried out the analysis of mouse IgG3 antibody generation aspect.This analysis is specific to mouse IgG3, and in this was analyzed, other mouse IgG isotype was negative.
The result
The expression of chimeric PV1-IgG3: according to described in material and method and Figure 15, clone's V
LAnd V
HBe prepared into little exon (Figure 15), and it is attached in a kind of expression vector.Then, be among the NSO with this expression vector transfection to rat bone marrow tumour cell, to produce chimeric PV1-IgG3.With several transfectant exhausted substratum by elisa assay mouse IgG3 production of antibodies, and analyzed and the combining of EL4 cell with FACScan.In two kinds of analyses, most of transfectants are male.A kind of transfectant is selected owing to the antibody producing rate is high, in 1 liter of serum free medium with its enlarged culturing.By affinity chromatography, from 1 liter of exhausted substratum, be purified into PV1-IgG3.Productive rate＞10mg/l.
Embodiment 17 characterizes the chimeric PV1-IgG3 of purifying by HPLC and SDS-PAGE
Method
Proteic purifying: in 1 liter of Gibco serum-free hybridoma substratum, cultivate a kind of (cloning No. 1) in the high transfectant of IgG3 expression.When cell survival reaches 30% or when lower, results exhausted culture supernatant; And it is concentrated into 200ml, and with a PharmaciaP1 pump (2-3ml/ minute), on the albumen-A Sepharose post with its application of sample to one 5ml.Then, wash this post, use the described antibody of MgCl μ wash-out of 3.5M then with the PBS that contains extra 0.1M NaCl (final concentration of NaCl is 0.25M).Then, one with the PBS equilibrated PD-10 post that contains extra 0.1M NaCl on, make the albumen desalination of wash-out.Being stored in before 4 ℃,, filter and take off the protein solution of supersalt by the filter paper of a 0.2mm.The same with all mouse IgG3, high density (＞1mg/ml) down PV1-IgG3 precipitates when low temperature, by warmly then dissolving back in the solution at 37 ℃.In room temperature, this antibody then remains in the solution.As if the circulation repeatedly of cryoprecipitate do not influence the activity of the conjugated antigen of this antibody.
Carry out purity testing by size exclusion HPLC and SDS-PAGE: use the Perkin Elmer HPLC system that forms by a PEISS 200 Advanced LC Sample Processor (sample processor), a PE Series410Bio LC Pump (pump), a PE 235C Diode Array Detector (diode-array detector) and PE Nelson 600Series LINK, carry out size exclusion HPLC.Use Perkin Elmer Turbochrom NavigatorVersion 4.1 softwares, control automatic sampler, pump and detector; And with this software obtain, storage and processing data.With two TosoHaas TSK-GELG3000SWXL size exclusion HPLC posts (TosoHaas, catalog number (Cat.No.) 08541,7.8mm * 300mm, the granularity of 5mm, the pore size of 250 ) that are connected in series, finish separation.Moving phase is 200mM potassiumphosphate/150mM Repone K of pH6.9, and flow velocity is 1.00ml/ minute.Under 220nm wavelength and 280nm wavelength, monitor the elutriant of this post with spectrophotometer.The volume injected of undiluted PV1-IgG3 sample is 50 μ l (63.5 μ g).According to standard method, carry out SDS-PAGE.
The result
By size exclusion HPLC and SDS-PAGE, analyze the purity of isolating PV1-IgG3.The HPLC elution profile that in Figure 16, has shown PV1-IgG3.According to this analysis, this albumen is 99% monomer, and has the mobility that is equivalent to molecular weight 150kD.PV1, PV1-IgG3 and isotype also show the SDS-PAGE analysis that impinges upon under the non-reduced condition: all three kinds of antibody all have the molecular weight (Figure 17 A) of about 150kD.The more weak band of seeing in Figure 17 A is the artifact that seethes with excitement and form in not having the SDS of reductive action owing to described sample.They have reflected the number of uncompleted interchain disulfide bond in the described antibody.But (Figure 17 B) analyzes three kinds of same albumen and then show under reductive condition: PV1, rather than PV1-IgG3 or isotype contrast have the slightly high heavy chain of 50kD molecular weight of the common being seen IgG of molecular weight ratio.Therefore, perhaps hamster antibody PV1 at C
HAsn in 3
297Obtain the heavy chain glycosylation, perhaps there is an extra glycosylation site its other position in heavy chain.Discuss as later, this rare glycosylation pattern may impel the non-specific binding of PV1 and EL4 cell, perhaps is that the interaction by lectin/carbohydrate comes combination.
Embodiment 18
Method
Flow cytometry: at 4 ℃ of PV1,37.51 or PV1-IgG3 dyeing mouse T clone EL4 cell (2.5 * 10 with 1 μ g/ml
5Individual cell/0.2ml) reach 30 minutes washs them with the cold PBS of 2ml then, and puts together the specificity second antibody (10 μ g/ml) of fluorescence dye with its dyeing with 20 μ l.In the dark in 4 ℃ hatch 20 minutes after, wash described cell with PBS, and (Becton Dickenson, Milpitas analyze CA) with FACScan.
In described competitive assay, contrast the EL4 cell (2.5 * 10 that dyes in the dark with the R-PE-PV1 of 1 μ g/ml and PV1, PV1-IgG3 or the IgG3 isotype of 25 μ g/ml in 4 ℃
5Individual cell/0.2ml) reach 30 minutes; Wash described cell with PBS, and analyze with FACScan.Also carry out similar competitive assay with multi-form 145.2C11.In reversible competitive assay, in 4 ℃ of PV1 dyeing EL4 cells (2.5 * 10 with the PV1-IgG3 of 1 μ g/ml and 25 μ g/ml
5Individual cell/0.2ml) reach 30 minutes; Use the PBS washed twice, and with the anti-mouse IgG of the donkey of puting together FITC (H+L) with its dyeing, washing then, and analyze with FACScan.In order to control the non-specific binding of second antibody and PV1, with excessive PV1 the EL4 cell is not dyeed under the situation of PV1-IgG3 having, and analyze described cell.
As for mouse T cell dyeing, at 4 ℃ with the mouse IgG3 isotypes contrasts (FLOPC 21) of 1 μ g/ml or PV1-IgG3 splenocyte (2.5 * 10 to BALB/c mouse
5Individual cell/0.2ml) dyeing reaches 30 minutes; Put together the 145.2C11 (10 μ g/ml) of FITC and goat anti-mouse IgG 3 (the 10 μ g/ml) dyeing that 20 μ l put together R-PE then with the cold PBS washing of 2ml, and with 20 μ l.4 ℃ in dark, hatch 20 minutes after, wash described cell with PBS, and analyze with FACScan.
The result
Characterize PV1 and PV1-IgG3 by flow cytometry: making the CD28 positive T cell with PV1 is EL4 dyeing, and analyzes with FACScan.Painted mode shows: PV1 in two different sites in conjunction with EL4 cell (Figure 17 A).In addition, and PV1 and the anti-mouse T of several armenian hamster cell antibody (145.2C11, anti--CD3; H57-597, anti--TCR; And UC10-4F10-11, anti-CTLA 4), also combine (data not shown) with the negative myeloma cell line NS0 of CD28 non-specificly.On the contrary, Syria hamster anti--CD28 antibody 37.51 only with the EL4 cell on a locus specificity combine (Figure 17 B).It seems that PV1 except in conjunction with the CD28, is attached on other site also non-specificly; It may be interaction by carbohydrate/lectin type.As shown in Figure 17 C, chimeric PV1-IgG3 does not comprise the activity of this non-specific binding.This antibody combines with the EL4 cell in the mode that is similar to 37.51 combination, and the NS0 cell (data not shown) of its debond CD28 feminine gender.Therefore, the non-specific binding characteristic of PV1 depends on the CH of this distinct antibodies, and passes through packing interaction and eliminate this specific character.
In order to confirm that PV1-IgG3 comprises the CD28 specific binding activity, we have adopted the FACScan competition analysis.In these experiments, the PV1 that puts together R-PE is mixed with unlabelled PV1, PV1-IgG3 or the mouse IgG3 contrast of excessive (25 times), and make the EL4 cell dyeing with this mixture.As shown in Figure 18 A, PV1 and PV1-IgG3, rather than isotype contrast stop the PV1 that puts together R-PE to be attached on the EL4 cell.The inhibition that is caused by PV1-IgG3 is less than the inhibition that is caused by PV1, and we are interpreted as these data: PV1-IgG3 and the PV1 competition CD28 site of puting together R-PE, rather than compete non-specific site.Be that 145.2C11 (the anti-mouse CD3 of armenian hamster) and chimeric 145.2C11-IgG3 both stop the 145.2C11 that puts together R-PE to be attached to (Figure 18 B) on the EL4 cell similarly; But because this chimeric antibody can not be eliminated the non-specific binding of R-PE-145.2C11 and cell, thereby this antibody efficient is lower.
The PV1 that we also use excessive (25 times) and PV1-IgG3 competition combines with the EL4 cell, has done reversible competitive assay.Though do not have mark PV-1-IgG3 in this case, discern it specifically with the anti-mouse antibodies of the donkey of puting together FITC.Result among Figure 18 C shows: by excessive PV1 causes PV1-IgG3 and EL4 cell bonded are suppressed, almost be completely; Thereby confirmed that PV1 combines same epi-position with PV1-IgG3.
At last, with PV1-IgG3 mouse boosting cell is dyeed.The splenocyte that is surrounded by PV1-IgG3 is that the goat anti-mouse IgG 3 that second antibody is puted together R-PE is discerned specifically.Simultaneously, also the 145.2C11 that puts together FITC is added among the splenocyte, thus mark CD3 positive cell.In bi-color flow cytometry was analyzed, PV1-IgG3 made the CD3 positive cell dyeing specifically, and did not make CD3 negative cells dyeing (Figure 19 B).On the contrary, the contrast of mouse IgG3 isotype does not make CD3 positive cell painted (Figure 19 A).Therefore, chimeric PV1-IgG3 is identified in a kind of antigen of expressing on the mouse T cell, and this is the conjugated antigen activity that is consistent with anti-CD28 antibody.
Bringing out property of collagen protein is arthritic induces for embodiment 19
Method
In order to isopyknic CFA (Wako, Japan) emulsive 125 μ g ox CII (CollagenGijutsu Kenkyukai, Japan), at the substrate intradermal immune mouse of mouse tail.At the 21st day,,, come the booster immunization mouse by the intradermal injection with ox CII 125 μ g, among the CFA.After initial immunity, with 1mg/kg/ days dosage, ooze the anti-CD28 antibody of pump continuous infusion (PV1-IgG3) by means of grade and reach 7 days, treat mouse.Arthritic generation by observing four pawls, is checked in after the immunity second time the 11st day; And according to before described (Tada, Y., A.Ho, D.-R.Koh, T.W.Mak.1996.J.Immunol.156:4520 .Tada, Y., A.Ho, T.Matsuyama T.W.Mak.1997.J.Exp.Med.185:231), comments grade with the inflammation of four pawls by 0 to 3 grade.Evaluate the grade of every pawl, and with four score additions; Therefore the top score of every mouse is 12.By remove the PTS of experiment mice with total mice, calculate arthritis index.
The result
With ox CII immunity mouse, observe the situation that mouse arthritis takes place.After the immunity second time the 11st day, in the mouse with anti-CD28 Antybody therapy, arthritis index (0.63 ± 0.50) (P＜0.01) is compared with contrast (7.50 ± 0.66) and is significantly reduced.
Method
Mouse: animal
From Charles River Japan, (Yokohama Japan), obtains female BALB/c mouse and C3H mouse to Inc..Animal is all closed the septulum with filtrated air of supporting in the pathogen-free domestic facility in cage, and can freely obtain food and water.When beginning to test, all mouse were 6 to 8 ages in week.
Antibody:
The reticent anti-mouse CD28 of type (PV1-IgG3) has the identical specificity with the PV-1 clone, but it does not have a strong agonist activity (Fc → IgG3) external.Anti-mouse CD154 (TRAP1, IgG1) available from BD PharMingen (San Diego, CA).CTLA4-Ig (CTLA-4/Fc mosaic) available from Genzyme (Cambridge, MA).
The tail skin grafting dermepenthesis:
To be transplanted to the dorsal part chest of acceptor mouse (C3H:H-2b) from the full thickness skin graft (0.5cm2) of donor mouse (BALB/c:H-2d) afterbody, and be fixed 7 days with first-aid dressing.Then, by visual observation every day, follow the tracks of the survival of graft.The epidermic grafting fabric texture of repelling the work that is defined as forfeiture＞80%.Multiple comparisons check carrying out statistical analysis with Dunnett.The numerical value of p＜0.05 is considered to significance.
Treatment plan:
With the anti-mouse CD28 of reticent type of 10 μ g, 50 μ g, 250 μ g, treat the acceptor of skin graft.Transplanting the same day (0 day) and behind hand the 3rd day and the 6th day, intraperitoneal gives anti-mouse CD154 of 250 μ g and 100 μ gCTLA4-Ig.
The result:
The T cell of blocking CD40 and CD28 by giving anti-mouse CD28 of reticent type and anti-mouse CD154 simultaneously stimulates approach jointly, then promotes the survival of the skin homotransplant in the C3H mouse effectively.Control animal repelled its graft at the 9th day.Independent anti-CD 40 L monoclonal antibody prolongs the survival (MST is 10 days) of homotransplant moderately, but when uniting with CD28, then observes and improve survival significantly, and half survival time (MST) was extended to above the 33rd day.This strategy obviously is not too effective when giving CTLA4-Ig and anti-mouse CD40L monoclonal antibody, and MST is 12 days.
The Fab of embodiment 21 anti-CD28 antibody and F (ab ') 2 segmental preparations
The segmental preparation of the Fab of anti-CD28 antibody
With immobilized ficin (Pierce, USA) digestion anti-people CD28 antibody (HuTN228).50mMTris-HCl pH6.8 damping fluid with containing 5mM EDTA and 11.5mM halfcystine hydrochloric acid activates immobilized ficin; And fill it in the post.Antibody-solutions is added on this post, and reach 2 or 3 days 37 ℃ of insulations.Wash this post with PBS, and, concentrate described Digestive system by ultrafiltration.With spissated Digestive system be added to gel-filtration column (TSKgel-3000SWxl, Tosoh, Japan) on, and collect suitable fraction, and concentrate by ultrafiltration.(, Abs280=1.4) determine proteic concentration according to absorbancy for 1mg/ml at 280nm; And confirm segmental size by SDS-PAGE.
The F of anti-CD28 antibody (ab ') 2 segmental preparations
With the method the same (except the concentration (1.15mM) and soaking time (spending the night once) of halfcystine), prepare anti-people CD28 antibody with Fab produced in fragments method.
Obviously, according to foregoing, may there be many improvement of the present invention and variation.Therefore must understand: in the scope of appending claims, can implement the present invention with the alternate manner except that this paper is concrete described.
All publications mentioned in this article, patent application, patent and other reference all are attached to herein by reference.
Sequence table
<110>VASQUEZ.Maximiliano
HINTON.Paul
TAMURA，Kouiehi
HIGASHI，Yauyuki
SEKI.Nobuo
UEDA，Hirotsugu
TSO，J.Yun
＜120〉anti-CD28 antibody of reticent type and application thereof
<130>200071USOPROV
<150>US?60/255,155
<151>2000-12-14
<160>21
<170>PatentIn?version?3.1
<210>1
<211>427
<212>DNA
＜213〉heterozygosis
<220>
<221>CDS
<222>(12)..(404)
<223>
<400>1
tctagaccac?c?atg?gag?tca?gac?aca?ctc?ctg?cta?tgg?gtg?ctg?ctg?ctc 50
Met?Glu?Ser?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu
1 5 10
tgg?gtt?cca?ggc?tcc?act?ggt?gac?att?gtg?ctc?acc?caa?tct?cca?gct 98
Trp?Val?Pro?Gly?Ser?Thr?Gly?Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala
15 20 25
tct?ttg?gct?gtg?tct?ctg?ggg?cag?aga?gcc?acc?atc?tcc?tgc?aga?gcc 146
Ser?Leu?Ala?Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Ser?Cys?Arg?Ala
30 35 40 45
agt?gaa?agt?gtt?gaa?tat?tat?gtc?aca?agt?tta?atg?cag?tgg?tac?caa 194
Ser?Glu?Ser?Val?Glu?Tyr?Tyr?Val?Thr?Ser?Leu?Met?Gln?Trp?Tyr?Gln
50 55 60
cag?aaa?cca?gga?cag?cca?ccc?aaa?ctc?ctc?atc?tat?gct?gca?tcc?aac 242
Gln?Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Asn
65 70 75
gta?gat?tct?ggg?gtc?cct?gcc?agg?ttt?agt?ggc?agt?ggg?tct?ggg?aca 290
Val?Asp?Ser?Gly?Val?Pro?Ala?Arg?PheSer?Gly?Ser?Gly?Ser?Gly?Thr
80 85 90
gac?ttc?agc?ctc?aac?atc?cat?cct?gtg?gag?gag?gat?gat?att?gca?atg 338
Asp?Phe?Ser?Leu?Asa?Ile?His?Pro?Val?Glu?Glu?Asp?Asp?Ile?Ala?Met
95 100 105
tat?ttc?tgt?cag?caa?agt?agg?aag?gtt?cca?ttc?acg?ttc?ggc?tcg?ggg 386
Tyr?Phe?Cys?Gln?Gln?Ser?Arg?Lys?Val?Pro?Phe?Thr?Phe?Gly?Ser?Gly
110 115 120 125
aca?aag?ttg?gaa?ata?aaa?cgtaagtaga?cttttgctct?aga 427
Thr?Lys?Leu?Glu?Ile?Lys
130
<210>2
<211>131
<212>PRT
＜213〉heterozygosis
<400>2
Met?Glu?Ser?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro
1 5 10 15
Gly?Ser?Thr?Gly?Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Ser?Leu?Ala
20 25 30 Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Ser?Cys?Arg?Ala?Ser?Glu?Ser
35 40 45
Val?Glu?Tyr?Tyr?Val?Thr?Ser?Leu?Met?Gln?Trp?Tyr?Gln?Gln?Lys?Pro
50 55 60
Gly?Gln?Pro?Pro?Lys?Leu?Leu?IIe?Tyr?Ala?Ala?Ser?Ash?Val?Asp?Ser
65 70 75 80
Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser
85 90 95
Leu?Asn?Ile?His?Pro?Val?Glu?Glu?Asp?Asp?Ile?Ala?Met?Tyr?Phe?Cys
100 105 110
Gln?Gln?Ser?Arg?Lys?Val?Pro?Phe?Thr?Phe?Gly?Ser?Gly?Thr?Lys?Leu
115 120 125
Glu?Ile?Lys
130
<210>3
<211>877
<212>DNA
＜213〉heterozygosis
<220>
<221>CDS
<222>(12)..(431)
<223>
<400>3
tctagaccac?c?atg?gag?tca?gac?aca?ctc?ctg?cta?tgg?gtg?ctg?ctg?ctc 50
Met?Glu?Ser?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu
1 5 10
tgg?gtt?cca?ggc?tcc?act?ggt?gac?att?gtg?ctc?acc?caa?tct?cca?gct 98
Trp?Val?Pro?Gly?Ser?Thr?Gly?Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala
15 20 25
tct?ttg?gct?gtg?tct?ctg?ggg?cag?aga?gcc?acc?atc?tcc?tgc?aga?gcc 146
Ser?Leu?Ala?Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Ser?Cys?Arg?Ala
30 35 40 45
agt?gaa?agt?gtt?gaa?tat?tat?gtc?aca?agt?tta?atg?cag?tgg?tac?caa 194
Ser?Glu?Ser?Val?Glu?Tyr?Tyr?Val?Thr?Ser?Leu?Met?Gln?Trp?Tyr?Gln
50 55 60
cag?aaa?cca?gga?cag?cca?ccc?aaa?ctc?ctc?atc?tat?gct?gca?tcc?aac 242
Gln?Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Asn
65 70 75
gta?gat?tct?ggg?gtc?cct?gcc?agg?ttt?agt?ggc?agt?ggg?tct?ggg?aca 290
Val?Asp?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr
80 85 90
gac?ttc?agc?ctc?aac?atc?cat?cct?gtg?gag?gag?gat?gat?att?gca?atg 338
Asp?Phe?Ser?Leu?Asn?Ile?His?Pro?Val?Glu?Glu?Asp?Asp?Ile?Ala?Met
95 100 105
tat?ttc?tgt?cag?caa?agt?agg?aag?gtt?cca?ttc?acg?ttc?ggc?tcg?ggg 386
Tyr?Phe?Cys?Gln?Gln?Ser?Arg?Lys?Val?Pro?Phe?Thr?Phe?Gly?Ser?Gly
110 115 120 125
aca?aag?ttg?gaa?ata?aaa?cgt?aag?tag?act?ttt?gct?cta?gat?cta 431
Thr?Lys?Leu?Glu?Ile?Lys?Arg?Lys Thr?Phe?Ala?Leu?Asp?Leu
130 135
131
gaccaccatg?gctgtcctgg?tgctgttcct?ctgcctggtt?gcatttccaa?gctgtgtcct 491
gtcccaggtg?cagctgaagg?agtcaggacc?tggcctggtg?gcgccctcac?agagcctgtc 551
catcacttgc?actgtctctg?gattttcatt?aaccagctat?ggtgtacact?gggttcgcca 611
gcctccagga?aagggtctgg?aatggctggg?agtcatatgg?cctggtggag?gcacaaattt 671
taattcggct?ctcatgtcca?gactgagcat?cagcgaagac?aactccaaga?gccaagtttt 731
cttaaaaatg?aacactctgc?aaactgatga?cacagccata?tattattgtg?ccagagatcg 791
ggcgtatggt?aactacctct?atgccatgga?ctactggggt?caaggaacct?cagtcaccgt 851
ctcctcaggt?aagaatggcc?tctaga 877
<210>4
<211>133
<212>PRT
＜213〉heterozygosis
<400>4
Met?Glu?Ser?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro
1 5 10 15
Gly?Ser?Thr?Gly?Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Ser?Leu?Ala
20 25 30
Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Ser?Cys?Arg?Ala?Ser?Glu?Ser
35 40 45
Val?Glu?Tyr?Tyr?Val?Thr?Ser?Leu?Met?Gln?Trp?Tyr?Gln?Gln?Lys?Pro
50 55 60
Gly?Gln?Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Asn?Val?Asp?Ser
65 70 75 80
Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser
85 90 95
Leu?Asn?Ile?His?Pro?Val?Glu?Glu?Asp?Asp?Ile?Ala?Met?Tyr?Phe?Cys
100 105 110
Gln?Gln?Ser?Arg?Lys?Val?Pro?Phe?Thr?Phe?Gly?Ser?Gly?Thr?Lys?Leu
115 120 125
Glu?Ile?Lys?Arg?Lys
130
<210>5
<211>6
<212>PRT
＜213〉heterozygosis
<400>5
Thr?Phe?Ala?Leu?Asp?Leu
1 5
<210>6
<211>450
<212>DNA
＜213〉heterozygosis
<220>
<221>CDS
<222>(12)..(431)
<223>
<400>6
tctagaccac?c?atg?gct?gtc?ctg?gtg?ctg?ttc?ctc?tgc?ctg?gtt?gca?ttt 50
Met?Ala?Val?Leu?Val?Leu?Phe?Leu?Cys?Leu?Val?Ala?Phe
1 5 10
cca?agc?tgt?gtc?ctg?tcc?cag?gtg?cag?ctg?cag?gag?tca?gga?cct?ggc 98
Pro?Ser?Cys?Val?Leu?Ser?Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly
15 20 25
ctg?gtg?aag?ccc?tca?gag?acc?ctg?tcc?ctc?act?tgc?gct?gtc?tct?gga 146
Leu?Val?Lys?Pro?Ser?Glu?Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Ser?Gly
30 35 40 45
ttt?tca?tta?acc?agc?tat?ggt?gta?cac?tgg?att?cgc?cag?cct?cca?gga 194
Phe?Ser?Leu?Thr?Ser?Tyr?Gly?Val?His?Trp?Ile?Arg?Gln?Pro?Pro?Gly
50 55 60
aag?ggt?ctg?gaa?tgg?ctg?gga?gtc?ata?tgg?cct?ggt?gga?ggc?aca?aat 242
Lys?Gly?Leu?Glu?Trp?Leu?Gly?Val?Ile?Trp?Pro?Gly?Gly?Gly?Thr?Asn
65 70 75
ttt?aat?tcg?gct?ctc?atg?tcc?aga?ctg?acc?atc?agc?gaa?gac?acc?tcc 290
Phe?Asn?Ser?Ala?Leu?Met?Ser?Arg?Leu?Thr?Ile?Ser?Glu?Asp?Thr?Ser
80 85 90
aag?aac?caa?gtt?tcc?tta?aaa?ttg?agc?tct?gtg?aca?gct?gct?gac?aca 338
Lys?Asn?Gln?Val?Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr
95 100 105
gcc?gta?tat?tat?tgt?gcc?aga?gat?cgg?gcg?tat?ggt?aac?tac?ctc?tat 386
Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Arg?Ala?Tyr?Gly?Asn?Tyr?Leu?Tyr
110 115 120 125
gcg?atg?gac?tac?tgg?ggt?caa?gga?acc?tta?gtc?acc?gtc?tcc?tca 431
Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
130 135 140
ggtaagaatg?gcctctaga 450
<210>7
<211>140
<212>PRT
＜213〉heterozygosis
<400>7
Met?Ala?Val?Leu?Val?Leu?Phe?Leu?Cys?Leu?Val?Ala?Phe?Pro?Ser?Cys
1 5 10 15
Val?Leu?Ser?Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys
20 25 30
Pro?Ser?Glu?Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Ser?Gly?Phe?Ser?Leu
35 40 45
Thr?Ser?Tyr?Gly?Val?His?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu
50 55 60
Glu?Trp?Leu?Gly?Val?Ile?Trp?Pro?Gly?Gly?Gly?Thr?Asn?Phe?Asn?Ser
65 70 75 80
Ala?Leu?Met?Ser?Arg?Leu?Thr?Ile?Ser?Glu?Asp?Thr?Ser?Lys?Asn?Gln
85 90 95
Val?Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr
100 105 110
Tyr?Cys?Ala?Arg?Asp?Arg?Ala?Tyr?Gly?Asn?Tyr?Leu?Tyr?Ala?Met?Asp
115 120 125
Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
130 135 140
<210>8
<211>427
<212>DNA
＜213〉heterozygosis
<220>
<221>CDS
<222>(12)..(404)
<223>
<400>8
tctagaccac?c?atg?gag?tca?gac?aca?ctc?ctg?cta?tgg?gtg?ctg?ctg?ctc 50
Met?Glu?Ser?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu
1 5 10
tgg?gtt?cca?ggc?tcc?act?ggt?gac?att?cag?atg?acc?caa?tct?cca?tct 98
Trp?Val?Pro?Gly?Ser?Thr?Gly?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser
15 20 25
tct?ttg?tct?gcg?tct?gtg?ggg?gac?agg?gtc?acc?atc?aca?tgc?aga?gcc 146
Ser?Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala
30 35 40 45
agt?gaa?agt?gtt?gaa?tat?tat?gtc?aca?agt?tta?atg?cag?tgg?tac?caa 194
Ser?Glu?Ser?Val?Glu?Tyr?Tyr?Val?Thr?Ser?Leu?Met?Gln?Trp?Tyr?Gln
50 55 60
cag?aaa?cca?gga?aag?gca?ccc?aaa?ctc?ctc?atc?tat?gct?gca?tcc?aac 242
Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Asn
65 70 75
gta?gat?tct?ggg?gtc?cct?tcc?agg?ttt?agt?ggc?agt?ggg?tct?ggg?aca 290
Val?Asp?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr
80 85 90
gac?ttc?acc?ctc?acc?atc?tct?tct?ctg?cag?ccg?gag?gat?att?gca?acg 338
Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr
95 100 105
tat?tac?tgt?cag?caa?agt?agg?aag?gtt?cca?ttc?acg?ttc?ggc?ggg?ggg 386
Tyr?Tyr?Cys?Gln?Gln?Ser?Arg?Lys?Val?Pro?Phe?Thr?Phe?Gly?Gly?Gly
110 115 120 125
aca?aag?gtg?gaa?ata?aaa?cgtaagtaga?cttttgctct?aga 427
Thr?Lys?Val?Glu?Ile?Lys
130
<210>9
<211>131
<212>PRT
＜213〉heterozygosis
<400>9
Met?Glu?Ser?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro
1 5 10 15
Gly?Ser?Thr?Gly?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser
20 25 30
Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Glu?Ser
35 40 45
Val?Glu?Tyr?Tyr?Val?Thr?Ser?Leu?Met?Gln?Trp?Tyr?Gln?Gln?Lys?Pro
50 55 60
Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Asn?Val?Asp?Ser
65 70 75 80
Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr
85 90 95
Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys
100 105 110
Gln?Gln?Ser?Arg?Lys?Val?Pro?Phe?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val
115 120 125
Glu?Ile?Lys
130
<210>10
<211>445
<212>DNA
＜213〉heterozygosis
<220>
<221>CDS
<222>(21)..(419)
<223>
<400>10
tctagacagt?ggggaacaat?atg?gat?tca?cag?atc?cag?gtc?ctc?atg?tcc?ctg 53
Met?Asp?Ser?Gln?Ile?Gln?Val?Leu?Met?Ser?Leu
1 5 10
ctc?ctc?tgg?atg?tct?ggt?gcc?tgt?gga?gat?att?gtg?atg?acc?cag?tct 101
Leu?Leu?Trp?Met?Ser?Gly?Ala?Cys?Gly?Asp?Ile?Val?Met?Thr?Gln?Ser
15 20 25
cca?tat?tcc?ctg?gct?gtg?tca?gca?gga?gag?aag?gtc?acc?atg?agt?tgc 149
Pro?Tyr?Ser?Leu?Ala?Val?Ser?Ala?Gly?Glu?Lys?Val?Thr?Met?Ser?Cys
30 35 40
agg?tcc?agt?cag?agc?ctc?tat?tac?agt?gga?atc?aaa?aag?aac?ctc?ttg 197
Arg?Ser?Ser?Gln?Ser?Leu?Tyr?Tyr?Ser?Gly?Ile?Lys?Lys?Asn?Leu?Leu
45 50 55
gcc?tgg?tac?cag?cag?aaa?cca?ggc?cag?tct?ccg?aaa?ctg?ctg?atc?tac 245
Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Lys?Leu?Leu?Ile?Tyr
60 65 70 75
ttt?aca?tct?act?cgg?tta?cct?ggg?gta?ccg?gat?cgc?ttc?aca?ggc?agt 293
Phe?Thr?Ser?Thr?Arg?Leu?Pro?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly?Ser
80 85 90
gga?tct?ggg?aca?gat?tac?act?ctc?acc?atc?acc?agt?gtc?cag?gct?gaa 341
Gly?Ser?Gly?Thr?Asp?Tyr?Thr?Leu?Thr?Ile?Thr?Ser?Val?Gln?Ala?Glu
95 100 105
gac?atg?ggg?cat?tat?ttc?tgt?cag?cag?ggt?ata?agc?act?ccg?ctc?acg 389
Asp?Met?Gly?His?Tyr?Phe?Cys?Gln?Gln?Gly?Ile?Ser?Thr?Pro?Leu?Thr
110 115 120
ttc?ggt?gat?ggc?acc?aag?ctg?gag?ata?aga?cgtaagtaga?atccaaagtc 439
Phe?Gly?Asp?Gly?Thr?Lys?Leu?Glu?Ile?Arg
125 130
tctaga 445
<210>11
<211>133
<212>PRT
＜213〉heterozygosis
<400>11
Met?Asp?Ser?Gln?Ile?Gln?Val?Leu?Met?Ser?Leu?Leu?Leu?Trp?Met?Ser
1 5 10 15
Gly?Ala?Cys?Gly?Asp?Ile?Val?Met?Thr?Gln?Ser?Pro?Tyr?Ser?Leu?Ala
20 25 30
Val?Ser?Ala?Gly?Glu?Lys?Val?Thr?Met?Ser?Cys?Arg?Ser?Ser?Gln?Ser
35 40 45
Leu?Tyr?Tyr?Ser?Gly?Ile?Lys?Lys?Asn?Leu?Leu?Ala?Trp?Tyr?Gln?Gln
50 55 60
Lys?Pro?Gly?Gln?Ser?Pro?Lys?Leu?Leu?Ile?Tyr?Phe?Thr?Ser?Thr?Arg
65 70 75 80
Leu?Pro?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly?Ser?Gly?Ser?Gly?Thr?Asp
85 90 95
Tyr?Thr?Leu?Thr?Ile?Thr?Ser?Val?Gln?Ala?Glu?Asp?Met?Gly?His?Tyr
100 105 110
Phe?Cys?Gln?Gln?Gly?Ile?Ser?Thr?Pro?Leu?Thr?Phe?Gly?Asp?Gly?Thr
115 120 125
Lys?Leu?Glu?Ile?Arg
130
<210>12
<211>467
<212>DNA
＜213〉heterozygosis
<220>
<221>CDS
<222>(16)..(444)
<223>
<400>12
tctagagtct?tcacc?atg?gta?tgg?ggc?ttg?atc?atc?atc?ttc?ctg?gtc?aca 51
Met?Val?Trp?Gly?Leu?Ile?Ile?Ile?Phe?Leu?Val?Thr
1 5 10
gca?gct?aca?ggt?gtc?cac?tcc?cag?gtc?cag?ttg?aag?cag?tct?ggg?gct 99
Ala?Ala?Thr?Gly?Val?His?Ser?Gln?Val?Gln?Leu?Lys?Gln?Ser?Gly?Ala
15 20 25
gag?ctt?gtg?aag?cct?gga?gcc?tca?gtg?aag?ata?tcc?tgc?aaa?act?tca 147
Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Thr?Ser
30 35 40
ggc?tat?acc?ttc?act?gat?ggc?tac?atg?aac?tgg?gtt?gag?cag?aag?cct 195
Gly?Tyr?Thr?Phe?Thr?Asp?Gly?Tyr?Met?Asn?Trp?Val?Glu?Gln?Lys?Pro
45 50 55 60
ggg?cag?ggc?ctt?gag?tgg?att?gga?aga?att?gat?cct?gat?agt?ggt?aat 243
Gly?Gln?Gly?Leu?Glu?Trp?Ile?Gly?Arg?Ile?Asp?Pro?Asp?Ser?Gly?Asn
65 70 75
act?cgg?tac?aat?cag?aaa?ttc?cag?ggc?aag?gcc?aca?ctg?act?aga?gac 291
Thr?Arg?Tyr?Asn?Gln?Lys?Phe?Gln?Gly?Lys?Ala?Thr?Leu?Thr?Arg?Asp
80 85 90
aaa?tcc?tcc?agc?aca?gtc?tac?atg?gac?ctc?agg?agc?ctg?aca?tct?gag 339
Lys?Ser?Ser?Ser?Thr?Val?Tyr?Met?Asp?Leu?Arg?Ser?Leu?Thr?Ser?Glu
95 100 105
gac?tct?gct?gtc?tat?tac?tgt?gcg?aga?gat?ggg?acc?ttc?tac?ggt?acc 387
Asp?Ser?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Gly?Thr?Phe?Tyr?Gly?Thr
110 115 120
tac?ggc?tac?tgg?tac?ttc?gat?ttc?tgg?ggc?cag?ggg?acc?cag?gtc?acc 435
Tyr?Gly?Tyr?Trp?Tyr?Phe?Asp?Phe?Trp?Gly?Gln?Gly?Thr?Gln?Val?Thr
125 130 135 140
gtc?tcc?tca?ggtgagtcct?taaaacctct?aga 467
Val?Ser?Ser
<210>13
<211>143
<212>PRT
＜213〉heterozygosis
<400>13
Met?Val?Trp?Gly?Leu?Ile?Ile?Ile?Phe?Leu?Val?Thr?Ala?Ala?Thr?Gly
1 5 10 15
Val?His?Ser?Gln?Val?Gln?Leu?Lys?Gln?Ser?Gly?Ala?Glu?Leu?Val?Lys
20 25 30
Pro?Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Thr?Ser?Gly?Tyr?Thr?Phe
35 40 45
Thr?Asp?Gly?Tyr?Met?Asn?Trp?Val?Glu?Gln?Lys?Pro?Gly?Gln?Gly?Leu
50 55 60
Glu?Trp?Ile?Gly?Arg?Ile?Asp?Pro?Asp?Ser?Gly?Asn?Thr?Arg?Tyr?Asn
65 70 75 80
Gln?Lys?Phe?Gln?Gly?Lys?Ala?Thr?Leu?Thr?Arg?Asp?Lys?Ser?Ser?Ser
85 90 95
Thr?Val?Tyr?Met?Asp?Leu?Arg?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val
100 105 110
Tyr?Tyr?Cys?Ala?Arg?Asp?Gly?Thr?Phe?Tyr?Gly?Thr?Tyr?Gly?Tyr?Trp
115 120 125
Tyr?Phe?Asp?Phe?Trp?Gly?Gln?Gly?Thr?Gln?Val?Thr?Val?Ser?Ser
130 135 140
<210>14
<211>46
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>14
tatagagctc?aagcttggat?ggtgggaaga?tggatacagt?tggtgc 46
<210>15
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>15
tatagagctc?aagcttccag?tggatagacc?gatggggctg?tcgttttggc 50
<210>16
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>16
tatagagctc?aagcttccag?tggatagaca?gatgggggtg?ttgttttggc 50
<210>17
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>17
tatagagctc?aagcttccag?tggatagacc?gttggggctg?tcgttttggc 50
<210>18
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>18
tatagagctc?aagcttccag?tggatagacc?gatggggctg?tcgttttggc 50
<210>19
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>19
tatagagctc?aagcttccag?tggatagacc?gatgggggtg?ttgttttggc 50
<210>20
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>20
tatagagctc?aagcttccag?tggatagtcc?gatggggctg?tcgttttggc 50
<210>21
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic DNA
<400>21
tatagagctc?cacttccagt?gccc 24
Claims (13)
1. anti-CD28 antibody of reticent type, it has: have the variable region of heavy chain and the variable region of light chain with aminoacid sequence among the SEQ ID NO:2 of aminoacid sequence among the SEQ ID NO:4, perhaps have the variable region of heavy chain and the variable region of light chain with aminoacid sequence among the SEQID NO:9 of aminoacid sequence among the SEQ ID NO:7; And has human IgG2 M3 CH.
2. the polynucleotide of the antibody of the claim 1 of encoding.
3. expression vector that comprises the polynucleotide of claim 2.
4. host cell that comprises the polynucleotide of claim 2.
5 one kinds of host cells that comprise the expression vector of claim 3.
6. method of producing the anti-CD28 antibody of reticent type, described method comprises:
Be suitable under the condition of described antibody expression, cultivating the host cell of claim 4; And from described culture, reclaim expressed antibody.
7. method of producing the anti-CD28 antibody of reticent type, described method comprises:
Be suitable under the condition of described antibody expression, cultivating the host cell of claim 5; And from described culture, reclaim expressed antibody.
8. method of producing the anti-CD28 antibody of reticent type, described method comprises:
The polynucleotide of claim 2 are incorporated in the host cell;
Be suitable for cultivating described host cell under the condition of described antibody expression; And from described culture, reclaim expressed antibody.
9. method of producing the anti-CD28 antibody of reticent type, described method comprises:
The expression vector of claim 3 is incorporated in the host cell;
Be suitable for cultivating described host cell under the condition of described antibody expression; And from described culture, reclaim expressed antibody.
10. medicinal compositions, described composition comprises anti-CD28 antibody of the reticent type of claim 1 and pharmaceutically acceptable composition.
11. the antibody of claim 1 is used for the treatment of application in the medicine that patient's intracorporeal organ or tissue transplantation repel in preparation.
12. the application of claim 11, wherein said medicine and another kind of immunosuppressive drug coupling.
13. antibody HuTN228, it has: have the variable region of heavy chain of aminoacid sequence among the SEQ ID NO:7, the variable region of light chain with aminoacid sequence among the SEQ ID NO:9 and human IgG2 M3 CH.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US25515500P | 2000-12-14 | 2000-12-14 | |
| US60/255,155 | 2000-12-14 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1489473A CN1489473A (en) | 2004-04-14 |
| CN1272345C true CN1272345C (en) | 2006-08-30 |
Family
ID=22967074
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB018226361A Expired - Fee Related CN1272345C (en) | 2000-12-14 | 2001-12-14 | Silensed anti-CD 28 antibodies and use thereof |
Country Status (18)
| Country | Link |
|---|---|
| EP (1) | EP1341553A4 (en) |
| JP (1) | JP2004515243A (en) |
| KR (1) | KR20040020866A (en) |
| CN (1) | CN1272345C (en) |
| AR (1) | AR031924A1 (en) |
| AU (2) | AU2002226086C1 (en) |
| BR (1) | BR0116686A (en) |
| CA (1) | CA2432736A1 (en) |
| CZ (1) | CZ20031909A3 (en) |
| HU (1) | HUP0400697A3 (en) |
| IL (1) | IL156262A0 (en) |
| MX (1) | MXPA03005327A (en) |
| NO (1) | NO20032542L (en) |
| NZ (1) | NZ526569A (en) |
| PL (1) | PL363239A1 (en) |
| RU (1) | RU2261723C2 (en) |
| WO (1) | WO2002047721A1 (en) |
| ZA (1) | ZA200305384B (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080095774A1 (en) * | 2001-02-16 | 2008-04-24 | Wyeth | Agents and Methods for Specifically Blocking CD28-Mediated Signaling |
| US7531168B2 (en) | 2001-02-16 | 2009-05-12 | Genetics Institute Llc | Method for downmodulating immune response in type I diabetes |
| BRPI0611901A2 (en) | 2005-06-14 | 2012-08-28 | Amgen, Inc | composition, lyophilized kit and process for preparing a composition |
| FR2951176A1 (en) | 2009-10-09 | 2011-04-15 | Tcl Pharma | MONOVALENT LIGANDS OF THE HUMAN CD28 RECEPTOR |
| WO2011101791A1 (en) * | 2010-02-18 | 2011-08-25 | Tcl Pharma | Anti-cd28 humanized antibodies |
| AU2017345490B2 (en) | 2016-10-21 | 2022-07-07 | Amgen Inc. | Pharmaceutical formulations and methods of making the same |
| KR20210035805A (en) | 2018-06-15 | 2021-04-01 | 플래그쉽 파이어니어링 이노베이션스 브이, 인크. | Increased immune activity through regulation of postcellular signaling factors |
| WO2020183471A1 (en) * | 2019-03-14 | 2020-09-17 | Biond Biologics Ltd. | A method for immunosuppression |
| MA55805A (en) | 2019-05-03 | 2022-03-09 | Flagship Pioneering Innovations V Inc | METHODS OF MODULATING IMMUNE ACTIVITY |
| JP2023509359A (en) | 2019-12-17 | 2023-03-08 | フラグシップ パイオニアリング イノベーションズ ブイ，インコーポレーテッド | Combination anticancer therapy with inducers of iron-dependent cell degradation |
| CN116096906A (en) | 2020-06-29 | 2023-05-09 | 旗舰创业创新五公司 | Virus engineered to promote saenox delivery and use thereof in treating cancer |
| IL300666A (en) | 2020-08-19 | 2023-04-01 | Xencor Inc | Anti-cd28 compositions |
| KR20230165276A (en) | 2021-03-31 | 2023-12-05 | 플래그쉽 파이어니어링 이노베이션스 브이, 인크. | Thanotransmission polypeptides and their use in the treatment of cancer |
| EP4363059A1 (en) | 2021-06-29 | 2024-05-08 | Flagship Pioneering Innovations V, Inc. | Immune cells engineered to promote thanotransmission and uses thereof |
| WO2024040195A1 (en) | 2022-08-17 | 2024-02-22 | Capstan Therapeutics, Inc. | Conditioning for in vivo immune cell engineering |
| WO2024077191A1 (en) | 2022-10-05 | 2024-04-11 | Flagship Pioneering Innovations V, Inc. | Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6641809B1 (en) * | 1990-03-26 | 2003-11-04 | Bristol-Myers Squibb Company | Method of regulating cellular processes mediated by B7 and CD28 |
-
2001
- 2001-12-14 AR ARP010105828A patent/AR031924A1/en unknown
- 2001-12-14 NZ NZ526569A patent/NZ526569A/en unknown
- 2001-12-14 BR BR0116686-7A patent/BR0116686A/en not_active IP Right Cessation
- 2001-12-14 HU HU0400697A patent/HUP0400697A3/en unknown
- 2001-12-14 AU AU2002226086A patent/AU2002226086C1/en not_active Ceased
- 2001-12-14 CA CA002432736A patent/CA2432736A1/en not_active Abandoned
- 2001-12-14 CZ CZ20031909A patent/CZ20031909A3/en unknown
- 2001-12-14 CN CNB018226361A patent/CN1272345C/en not_active Expired - Fee Related
- 2001-12-14 KR KR10-2003-7007798A patent/KR20040020866A/en not_active Application Discontinuation
- 2001-12-14 MX MXPA03005327A patent/MXPA03005327A/en unknown
- 2001-12-14 EP EP01995504A patent/EP1341553A4/en not_active Withdrawn
- 2001-12-14 IL IL15626201A patent/IL156262A0/en unknown
- 2001-12-14 PL PL01363239A patent/PL363239A1/en not_active Application Discontinuation
- 2001-12-14 AU AU2608602A patent/AU2608602A/en active Pending
- 2001-12-14 WO PCT/US2001/047955 patent/WO2002047721A1/en not_active Application Discontinuation
- 2001-12-14 RU RU2003121231/15A patent/RU2261723C2/en not_active IP Right Cessation
- 2001-12-14 JP JP2002549291A patent/JP2004515243A/en not_active Withdrawn
-
2003
- 2003-06-05 NO NO20032542A patent/NO20032542L/en not_active Application Discontinuation
- 2003-07-11 ZA ZA200305384A patent/ZA200305384B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO20032542D0 (en) | 2003-06-05 |
| JP2004515243A (en) | 2004-05-27 |
| CZ20031909A3 (en) | 2003-11-12 |
| HUP0400697A2 (en) | 2004-06-28 |
| HUP0400697A3 (en) | 2007-05-02 |
| CN1489473A (en) | 2004-04-14 |
| IL156262A0 (en) | 2004-01-04 |
| RU2003121231A (en) | 2005-02-10 |
| EP1341553A1 (en) | 2003-09-10 |
| MXPA03005327A (en) | 2004-12-03 |
| EP1341553A4 (en) | 2004-07-28 |
| NO20032542L (en) | 2003-08-07 |
| RU2261723C2 (en) | 2005-10-10 |
| KR20040020866A (en) | 2004-03-09 |
| CA2432736A1 (en) | 2002-06-20 |
| ZA200305384B (en) | 2004-10-11 |
| AU2002226086C1 (en) | 2006-03-09 |
| PL363239A1 (en) | 2004-11-15 |
| NZ526569A (en) | 2005-07-29 |
| AU2002226086B2 (en) | 2005-08-25 |
| WO2002047721A1 (en) | 2002-06-20 |
| AR031924A1 (en) | 2003-10-08 |
| BR0116686A (en) | 2003-12-30 |
| AU2608602A (en) | 2002-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1293098C (en) | Concatameric immunoadhesion | |
| CN1187374C (en) | Humanized antibody specific for human 4-1BB and pharmaceutical compsn comprising same | |
| CN1272345C (en) | Silensed anti-CD 28 antibodies and use thereof | |
| CN1211123C (en) | Humanized antibodies against leukocyte adhesion molecule VLA-4 | |
| CN1271205C (en) | Reconstituted human anti-HM1.24 antibody | |
| CN1359302A (en) | Remedies for immunoogical diseases | |
| CN1146442C (en) | Lymphotoxin- | |
| CN1222162A (en) | Mutated nonactivating IgG2 domains and anti-cd3 antibodies incorporating same | |
| CN1898267A (en) | Modified anti-cd52 antibody | |
| CN1599754A (en) | Anti-TRAIL-R antibodies | |
| CN1155904A (en) | LAG-3 protein soluble polypeptide fractions, method of production, therapeutic composition and anti-idiotype antibody | |
| CN1878790A (en) | Neutralizable epitope of HGF and neutralizing antibody binding to the same | |
| CN1191855C (en) | Remedies for lymphocytic tumors | |
| CN1341124A (en) | Anti-CD3 immunotoxins and therapeutic use thereof | |
| CN1352650A (en) | Recombinant IL-18 antagonist useful in treatment of IL-18 mediated disorders | |
| CN1200737A (en) | Recombinant anti-CD4 antibodies for human therapy | |
| CN1076966A (en) | The clone of the humanized monoclonal antibodies of anti-human interleukin-4 and expression | |
| CN1620468A (en) | Novel anti-igf-ir antibodies and uses thereof | |
| CN1656122A (en) | Human monoclonal antibodies to interleukin-5 and methods and compositions comprising same | |
| CN1541224A (en) | Antagonistic anti-hTNFSF13b Human antibodies | |
| CN1277632A (en) | Natural human antibody | |
| CN1606453A (en) | Cripto blocking antibodies and uses thereof | |
| CN1571797A (en) | Anti-hla-dr antibody | |
| CN1439022A (en) | Human monoclonal antibody against a costimulatory signal transduction molecule AILIM and pharmaceutical use thereof | |
| CN1898264A (en) | Novel anti-il 13 antibodies for treating cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1064943 Country of ref document: HK |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1064943 Country of ref document: HK |