NZ616809B2 - Anti-b7-h3 antibody - Google Patents
Anti-b7-h3 antibody Download PDFInfo
- Publication number
- NZ616809B2 NZ616809B2 NZ616809A NZ61680912A NZ616809B2 NZ 616809 B2 NZ616809 B2 NZ 616809B2 NZ 616809 A NZ616809 A NZ 616809A NZ 61680912 A NZ61680912 A NZ 61680912A NZ 616809 B2 NZ616809 B2 NZ 616809B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- seq
- amino acid
- sequence represented
- antibody
- nucleotide
- Prior art date
Links
- 102000004965 antibodies Human genes 0.000 title claims abstract description 694
- 108090001123 antibodies Proteins 0.000 title claims abstract description 694
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 378
- 101700015421 CD276 Proteins 0.000 claims abstract description 247
- 102100019456 CD276 Human genes 0.000 claims abstract description 247
- 230000000694 effects Effects 0.000 claims abstract description 80
- 201000011510 cancer Diseases 0.000 claims abstract description 53
- 230000000259 anti-tumor Effects 0.000 claims abstract description 26
- 238000004519 manufacturing process Methods 0.000 claims abstract description 22
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 16
- 230000001419 dependent Effects 0.000 claims abstract description 14
- 206010057249 Phagocytosis Diseases 0.000 claims abstract description 13
- 230000008782 phagocytosis Effects 0.000 claims abstract description 13
- 230000001404 mediated Effects 0.000 claims abstract description 10
- 210000004027 cells Anatomy 0.000 claims description 319
- 229920001850 Nucleic acid sequence Polymers 0.000 claims description 236
- 150000001413 amino acids Chemical class 0.000 claims description 223
- 239000002773 nucleotide Substances 0.000 claims description 137
- 125000003729 nucleotide group Chemical group 0.000 claims description 137
- 239000000203 mixture Substances 0.000 claims description 50
- 230000014509 gene expression Effects 0.000 claims description 45
- 206010028980 Neoplasm Diseases 0.000 claims description 42
- 229940035295 Ting Drugs 0.000 claims description 33
- 239000008194 pharmaceutical composition Substances 0.000 claims description 23
- 229920000023 polynucleotide Polymers 0.000 claims description 22
- 239000002157 polynucleotide Substances 0.000 claims description 22
- 239000002253 acid Substances 0.000 claims description 21
- 230000004540 complement-dependent cytotoxicity Effects 0.000 claims description 15
- 230000035693 Fab Effects 0.000 claims description 14
- 230000000295 complement Effects 0.000 claims description 13
- 230000001472 cytotoxic Effects 0.000 claims description 11
- 230000002401 inhibitory effect Effects 0.000 claims description 10
- 206010038389 Renal cancer Diseases 0.000 claims description 8
- 201000010982 kidney cancer Diseases 0.000 claims description 8
- 230000004048 modification Effects 0.000 claims description 8
- 238000006011 modification reaction Methods 0.000 claims description 8
- 206010006187 Breast cancer Diseases 0.000 claims description 7
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 7
- 206010025650 Malignant melanoma Diseases 0.000 claims description 7
- 201000007270 liver cancer Diseases 0.000 claims description 7
- 201000005202 lung cancer Diseases 0.000 claims description 7
- 201000001441 melanoma Diseases 0.000 claims description 7
- 206010005003 Bladder cancer Diseases 0.000 claims description 6
- 206010033128 Ovarian cancer Diseases 0.000 claims description 6
- 208000008443 Pancreatic Carcinoma Diseases 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 6
- 201000011231 colorectal cancer Diseases 0.000 claims description 6
- 230000002860 competitive Effects 0.000 claims description 6
- 201000002528 pancreatic cancer Diseases 0.000 claims description 6
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 6
- 206010017758 Gastric cancer Diseases 0.000 claims description 5
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 5
- 230000001413 cellular Effects 0.000 claims description 5
- 230000002708 enhancing Effects 0.000 claims description 5
- 201000004101 esophageal cancer Diseases 0.000 claims description 5
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 5
- 230000001105 regulatory Effects 0.000 claims description 5
- 201000011549 stomach cancer Diseases 0.000 claims description 5
- 150000004676 glycans Chemical class 0.000 claims description 4
- 230000002611 ovarian Effects 0.000 claims description 4
- 231100000433 cytotoxic Toxicity 0.000 claims description 3
- 230000003013 cytotoxicity Effects 0.000 claims description 2
- 231100000135 cytotoxicity Toxicity 0.000 claims description 2
- 210000000481 Breast Anatomy 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 description 154
- 239000000427 antigen Substances 0.000 description 66
- 102000038129 antigens Human genes 0.000 description 66
- 108091007172 antigens Proteins 0.000 description 66
- 241000699666 Mus <mouse, genus> Species 0.000 description 63
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 59
- 239000004473 Threonine Substances 0.000 description 59
- 235000008521 threonine Nutrition 0.000 description 59
- 229960003767 Alanine Drugs 0.000 description 58
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 58
- 235000004279 alanine Nutrition 0.000 description 58
- 239000000243 solution Substances 0.000 description 52
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 51
- 239000004474 valine Substances 0.000 description 51
- MTCFGRXMJLQNBG-UWTATZPHSA-N D-serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-N 0.000 description 50
- 235000004400 serine Nutrition 0.000 description 50
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 49
- 235000005772 leucine Nutrition 0.000 description 49
- 125000000539 amino acid group Chemical group 0.000 description 48
- 210000004408 Hybridomas Anatomy 0.000 description 45
- 239000002609 media Substances 0.000 description 38
- 229920002676 Complementary DNA Polymers 0.000 description 34
- 102000004169 proteins and genes Human genes 0.000 description 34
- 108090000623 proteins and genes Proteins 0.000 description 34
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 33
- 235000018102 proteins Nutrition 0.000 description 33
- 239000002299 complementary DNA Substances 0.000 description 28
- 229960002743 Glutamine Drugs 0.000 description 27
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 27
- 239000004472 Lysine Substances 0.000 description 27
- 235000004554 glutamine Nutrition 0.000 description 27
- 229960003646 lysine Drugs 0.000 description 27
- 235000018977 lysine Nutrition 0.000 description 27
- 238000000034 method Methods 0.000 description 27
- 229960000310 ISOLEUCINE Drugs 0.000 description 26
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 26
- 108010045030 monoclonal antibodies Proteins 0.000 description 25
- 102000005614 monoclonal antibodies Human genes 0.000 description 25
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 24
- 229920001184 polypeptide Polymers 0.000 description 24
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 23
- 239000007759 RPMI Media 1640 Substances 0.000 description 22
- 239000012091 fetal bovine serum Substances 0.000 description 22
- 239000006228 supernatant Substances 0.000 description 22
- 239000004475 Arginine Substances 0.000 description 21
- 229960003121 arginine Drugs 0.000 description 21
- 235000009697 arginine Nutrition 0.000 description 21
- 239000000523 sample Substances 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 17
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 16
- 150000002500 ions Chemical class 0.000 description 16
- 238000000746 purification Methods 0.000 description 16
- 229940024606 Amino Acids Drugs 0.000 description 15
- 229940019746 Antifibrinolytic amino acids Drugs 0.000 description 15
- 229940021015 I.V. solution additive Amino Acids Drugs 0.000 description 15
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 15
- 210000002540 Macrophages Anatomy 0.000 description 15
- 239000003795 chemical substances by application Substances 0.000 description 15
- 230000036961 partial Effects 0.000 description 15
- 239000011780 sodium chloride Substances 0.000 description 15
- 206010035226 Plasma cell myeloma Diseases 0.000 description 14
- 108091006028 chimera Proteins 0.000 description 14
- 201000000050 myeloid neoplasm Diseases 0.000 description 14
- 210000000628 Antibody-Producing Cells Anatomy 0.000 description 12
- 102000004851 Immunoglobulin G Human genes 0.000 description 12
- 210000000952 Spleen Anatomy 0.000 description 12
- 239000012228 culture supernatant Substances 0.000 description 12
- 230000003053 immunization Effects 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- -1 probes Polymers 0.000 description 11
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 11
- 229960005190 Phenylalanine Drugs 0.000 description 11
- 241000700159 Rattus Species 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 239000003085 diluting agent Substances 0.000 description 10
- 239000000975 dye Substances 0.000 description 10
- 238000002649 immunization Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 230000004614 tumor growth Effects 0.000 description 10
- 241000588724 Escherichia coli Species 0.000 description 9
- 241000282412 Homo Species 0.000 description 9
- 230000002950 deficient Effects 0.000 description 9
- 108091007521 restriction endonucleases Proteins 0.000 description 9
- 230000001131 transforming Effects 0.000 description 9
- UCSJYZPVAKXKNQ-HZYVHMACSA-N 1-[(1S,2R,3R,4S,5R,6R)-3-carbamimidamido-6-{[(2R,3R,4R,5S)-3-{[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxan-2-yl]oxy}-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy}-2,4,5-trihydroxycyclohexyl]guanidine Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 102100019451 CD80 Human genes 0.000 description 8
- 101700080477 CD80 Proteins 0.000 description 8
- 229960002989 Glutamic Acid Drugs 0.000 description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 8
- 238000004166 bioassay Methods 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 8
- 230000000875 corresponding Effects 0.000 description 8
- 238000011033 desalting Methods 0.000 description 8
- 235000013922 glutamic acid Nutrition 0.000 description 8
- 239000004220 glutamic acid Substances 0.000 description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- 238000002372 labelling Methods 0.000 description 8
- 230000002194 synthesizing Effects 0.000 description 8
- 210000004881 tumor cells Anatomy 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 206010003445 Ascites Diseases 0.000 description 7
- 229960005261 Aspartic Acid Drugs 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 7
- 101710011976 PDCD1LG2 Proteins 0.000 description 7
- 102100007289 PDCD1LG2 Human genes 0.000 description 7
- 210000001744 T-Lymphocytes Anatomy 0.000 description 7
- 102000004142 Trypsin Human genes 0.000 description 7
- 108090000631 Trypsin Proteins 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 235000003704 aspartic acid Nutrition 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 229960001322 trypsin Drugs 0.000 description 7
- 239000012588 trypsin Substances 0.000 description 7
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- 238000001042 affinity chromatography Methods 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000000919 ceramic Substances 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 230000023298 conjugation with cellular fusion Effects 0.000 description 6
- 230000009089 cytolysis Effects 0.000 description 6
- 239000002502 liposome Substances 0.000 description 6
- 230000002934 lysing Effects 0.000 description 6
- 230000005700 syncytium formation by plasma membrane fusion Effects 0.000 description 6
- 101700072555 CLEA Proteins 0.000 description 5
- 241000725101 Clea Species 0.000 description 5
- 229940088598 Enzyme Drugs 0.000 description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 5
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 5
- 210000002966 Serum Anatomy 0.000 description 5
- 238000000692 Student's t-test Methods 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000011091 antibody purification Methods 0.000 description 5
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 230000001629 suppression Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 235000002374 tyrosine Nutrition 0.000 description 5
- 210000000683 Abdominal Cavity Anatomy 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 210000000088 Lip Anatomy 0.000 description 4
- 210000004698 Lymphocytes Anatomy 0.000 description 4
- 239000012515 MabSelect SuRe Substances 0.000 description 4
- 210000004379 Membranes Anatomy 0.000 description 4
- 108020004999 Messenger RNA Proteins 0.000 description 4
- 229920002873 Polyethylenimine Polymers 0.000 description 4
- 108020004412 RNA 3' Polyadenylation Signals Proteins 0.000 description 4
- 229960005322 Streptomycin Drugs 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 230000003115 biocidal Effects 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 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 4
- 238000003260 fluorescence intensity Methods 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 239000001963 growth media Substances 0.000 description 4
- 238000001114 immunoprecipitation Methods 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 4
- 239000012139 lysis buffer Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229920002106 messenger RNA Polymers 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- 229960000060 monoclonal antibodies Drugs 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 230000003248 secreting Effects 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 101710027066 ALB Proteins 0.000 description 3
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 3
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 3
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 3
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 3
- 229960001230 Asparagine Drugs 0.000 description 3
- 206010003816 Autoimmune disease Diseases 0.000 description 3
- 102100007290 CD274 Human genes 0.000 description 3
- 101710012053 CD274 Proteins 0.000 description 3
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- 210000001165 Lymph Nodes Anatomy 0.000 description 3
- 229940049954 Penicillin Drugs 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960000626 benzylpenicillin Drugs 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000003247 decreasing Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 3
- 230000002068 genetic Effects 0.000 description 3
- 201000010915 glioblastoma multiforme Diseases 0.000 description 3
- 230000001965 increased Effects 0.000 description 3
- 229940079866 intestinal antibiotics Drugs 0.000 description 3
- 210000004962 mammalian cells Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 3
- 108091008117 polyclonal antibodies Proteins 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 230000002829 reduced Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2R,3R,4S,5R,6S)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2S,3R,4S,5R,6R)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2R,3R,4S,5R,6R)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N (3β)-Cholest-5-en-3-ol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N 2-mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 2
- 229960003896 Aminopterin Drugs 0.000 description 2
- 229960003589 Arginine hydrochloride Drugs 0.000 description 2
- 208000006673 Asthma Diseases 0.000 description 2
- 210000004369 Blood Anatomy 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 210000000170 Cell Membrane Anatomy 0.000 description 2
- 210000001106 Chromosomes, Artificial, Yeast Anatomy 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N DEOXYTHYMIDINE Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 230000036947 Dissociation constant Effects 0.000 description 2
- 229940110715 ENZYMES FOR TREATMENT OF WOUNDS AND ULCERS Drugs 0.000 description 2
- 241000272184 Falconiformes Species 0.000 description 2
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N Hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 102000018358 Immunoglobulins Human genes 0.000 description 2
- 108060003951 Immunoglobulins Proteins 0.000 description 2
- 206010022114 Injury Diseases 0.000 description 2
- RCINICONZNJXQF-MZXODVADSA-N Intaxel Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 2
- 210000000822 Killer Cells, Natural Anatomy 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 210000004072 Lung Anatomy 0.000 description 2
- 101710028361 MARVELD2 Proteins 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 206010027476 Metastasis Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- LXCFILQKKLGQFO-UHFFFAOYSA-N Methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 229960001592 Paclitaxel Drugs 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102000007312 Recombinant Proteins Human genes 0.000 description 2
- 108010033725 Recombinant Proteins Proteins 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N Salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000000240 adjuvant Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000005377 adsorption chromatography Methods 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000139 costimulatory Effects 0.000 description 2
- 210000004748 cultured cells Anatomy 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000000539 dimer Substances 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
- 238000005516 engineering process Methods 0.000 description 2
- 229940071106 ethylenediaminetetraacetate Drugs 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 230000003899 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 229940020899 hematological Enzymes Drugs 0.000 description 2
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 2
- 229910052738 indium Inorganic materials 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 238000000302 molecular modelling Methods 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 230000003449 preventive Effects 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 239000012521 purified sample Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012146 running buffer Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 230000002269 spontaneous Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 108010064394 succinylated wheat germ agglutinin Proteins 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000004083 survival Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000001225 therapeutic Effects 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- NFGXHKASABOEEW-UHFFFAOYSA-N (+)-methoprene Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- ZMEWRPBAQVSBBB-GOTSBHOMSA-N (2S)-2-[[(2S)-2-[(2-aminoacetyl)amino]-3-(4-hydroxyphenyl)propanoyl]amino]-6-[[2-[2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetyl]amino]hexanoic acid Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC(=O)NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 ZMEWRPBAQVSBBB-GOTSBHOMSA-N 0.000 description 1
- OZRNSSUDZOLUSN-LBPRGKRZSA-M (4S)-4-[[4-[(2-amino-4-oxo-7,8-dihydro-1H-pteridin-6-yl)methylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate Chemical compound N=1C=2C(=O)NC(N)=NC=2NCC=1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-LBPRGKRZSA-M 0.000 description 1
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- BFFPVEVGHKMWLT-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;3,7-dihydropurin-6-one Chemical compound O=C1NC=NC2=C1NC=N2.O=C1NC(N)=NC2=C1NC=N2 BFFPVEVGHKMWLT-UHFFFAOYSA-N 0.000 description 1
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- 125000000094 2-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-Aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 1
- 102100001249 ALB Human genes 0.000 description 1
- 108060000679 ATG12 Proteins 0.000 description 1
- 101710034857 ATIC Proteins 0.000 description 1
- 210000001015 Abdomen Anatomy 0.000 description 1
- 229940028652 Abraxane Drugs 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N Ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- YBBLVLTVTVSKRW-UHFFFAOYSA-N Anastrozole Chemical compound N#CC(C)(C)C1=CC(C(C)(C#N)C)=CC(CN2N=CN=C2)=C1 YBBLVLTVTVSKRW-UHFFFAOYSA-N 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 210000000612 Antigen-Presenting Cells Anatomy 0.000 description 1
- BFYIZQONLCFLEV-DAELLWKTSA-N Aromasine Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC(=C)C2=C1 BFYIZQONLCFLEV-DAELLWKTSA-N 0.000 description 1
- 229940046844 Aromatase inhibitors Drugs 0.000 description 1
- 108090000206 Autoantibodies Proteins 0.000 description 1
- 102000003852 Autoantibodies Human genes 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 240000008371 Bacillus subtilis Species 0.000 description 1
- 229940075615 Bacillus subtilis Drugs 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 210000002469 Basement Membrane Anatomy 0.000 description 1
- 229960000686 Benzalkonium Chloride Drugs 0.000 description 1
- 108009000612 Bladder Cancer Proteins 0.000 description 1
- 210000004204 Blood Vessels Anatomy 0.000 description 1
- XOJVVFBFDXDTEG-UHFFFAOYSA-N Bute hydrocarbon Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 1
- 102000002110 C2 domain Human genes 0.000 description 1
- 108050009459 C2 domain Proteins 0.000 description 1
- QLNSPJMIYWEWNF-UHFFFAOYSA-N CC(C)CCCC(C)CCCC(C)CCCC(C)C.CC(C)CCCC(C)CCCC(C)CCCC(C)C Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C.CC(C)CCCC(C)CCCC(C)CCCC(C)C QLNSPJMIYWEWNF-UHFFFAOYSA-N 0.000 description 1
- 241000282832 Camelidae Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229960004562 Carboplatin Drugs 0.000 description 1
- OLESAACUTLOWQZ-UHFFFAOYSA-L Carboplatin Chemical compound O=C1O[Pt]([N]([H])([H])[H])([N]([H])([H])[H])OC(=O)C11CCC1 OLESAACUTLOWQZ-UHFFFAOYSA-L 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 238000003734 CellTiter-Glo Luminescent Cell Viability Assay Methods 0.000 description 1
- 210000001175 Cerebrospinal Fluid Anatomy 0.000 description 1
- 229960003260 Chlorhexidine Drugs 0.000 description 1
- 229940107161 Cholesterol Drugs 0.000 description 1
- 210000000349 Chromosomes Anatomy 0.000 description 1
- 210000003917 Chromosomes, Human Anatomy 0.000 description 1
- 230000036849 Clc Effects 0.000 description 1
- 210000001728 Clone Cells Anatomy 0.000 description 1
- 229920001405 Coding region Polymers 0.000 description 1
- 229940096422 Collagen Type I Drugs 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M Coomassie Brilliant Blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 210000004443 Dendritic Cells Anatomy 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000033147 ERVK-25 Human genes 0.000 description 1
- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Exidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N Gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- XLXSAKCOAKORKW-UHFFFAOYSA-N Gonadorelin Chemical class C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CCCN=C(N)N)NC(=O)C(CC(C)C)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 XLXSAKCOAKORKW-UHFFFAOYSA-N 0.000 description 1
- 108010069236 Goserelin Proteins 0.000 description 1
- BLCLNMBMMGCOAS-URPVMXJPSA-N Goserelin Chemical compound C([C@@H](C(=O)N[C@H](COC(C)(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NNC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 BLCLNMBMMGCOAS-URPVMXJPSA-N 0.000 description 1
- 229960002913 Goserelin Drugs 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 102100019441 ITGAM Human genes 0.000 description 1
- 101710006572 ITGAM Proteins 0.000 description 1
- 210000000987 Immune System Anatomy 0.000 description 1
- 108010027440 Immunoconjugates Proteins 0.000 description 1
- 102000018748 Immunoconjugates Human genes 0.000 description 1
- 102000004090 Immunoglobulin Isotypes Human genes 0.000 description 1
- 108090000539 Immunoglobulin Isotypes Proteins 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N Irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 241000282838 Lama Species 0.000 description 1
- 229940067606 Lecithin Drugs 0.000 description 1
- HPJKCIUCZWXJDR-UHFFFAOYSA-N Letrozole Chemical compound C1=CC(C#N)=CC=C1C(N1N=CN=C1)C1=CC=C(C#N)C=C1 HPJKCIUCZWXJDR-UHFFFAOYSA-N 0.000 description 1
- 108010000817 Leuprolide Proteins 0.000 description 1
- 229960004338 Leuprorelin Drugs 0.000 description 1
- 102100010665 MCM3AP Human genes 0.000 description 1
- 101710038091 MCM3AP Proteins 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 210000001616 Monocytes Anatomy 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 101710004849 NIP4-1 Proteins 0.000 description 1
- 108091007229 NSP3 Papain-like protease domain Proteins 0.000 description 1
- 208000002154 Non-Small-Cell Lung Carcinoma Diseases 0.000 description 1
- 108009000071 Non-small cell lung cancer Proteins 0.000 description 1
- 229940099990 Ogen Drugs 0.000 description 1
- 229920000272 Oligonucleotide Polymers 0.000 description 1
- 241000283898 Ovis Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 229950011098 PENDETIDE Drugs 0.000 description 1
- 102220396602 PET117 C57L Human genes 0.000 description 1
- 229940055729 Papain Drugs 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N Pathocidin Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108091005771 Peptidases Proteins 0.000 description 1
- 229940072417 Peroxidase Drugs 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108090000437 Peroxidases Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004696 Poly ether ether ketone Substances 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229940068977 Polysorbate 20 Drugs 0.000 description 1
- 229940068968 Polysorbate 80 Drugs 0.000 description 1
- 229940068965 Polysorbates Drugs 0.000 description 1
- 229960004063 Propylene glycol Drugs 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N Propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- GZUITABIAKMVPG-UHFFFAOYSA-N Raloxifene Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 GZUITABIAKMVPG-UHFFFAOYSA-N 0.000 description 1
- 229960004622 Raloxifene Drugs 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 206010072736 Rheumatic disease Diseases 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 108010070144 Single-Chain Antibodies Proteins 0.000 description 1
- 102000005632 Single-Chain Antibodies Human genes 0.000 description 1
- 108020004459 Small Interfering RNA Proteins 0.000 description 1
- 229940091252 Sodium supplements Drugs 0.000 description 1
- 229940075582 Sorbic Acid Drugs 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 210000002784 Stomach Anatomy 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 210000002437 Synoviocyte Anatomy 0.000 description 1
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 1
- 229960001603 Tamoxifen Drugs 0.000 description 1
- 210000004241 Th2 Cells Anatomy 0.000 description 1
- 229940104230 Thymidine Drugs 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 206010044412 Transitional cell carcinoma Diseases 0.000 description 1
- FPKOPBFLPLFWAD-UHFFFAOYSA-N Trinitrotoluene Chemical compound CC1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1[N+]([O-])=O FPKOPBFLPLFWAD-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K Trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940035504 Tromethamine Drugs 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 229960003048 Vinblastine Drugs 0.000 description 1
- HOFQVRTUGATRFI-XQKSVPLYSA-N Vinblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1N=C1[C]2C=CC=C1 HOFQVRTUGATRFI-XQKSVPLYSA-N 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Vitamin C Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 241000282485 Vulpes vulpes Species 0.000 description 1
- 102100018564 ZDHHC2 Human genes 0.000 description 1
- 101710024629 ZDHHC2 Proteins 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 229960002932 anastrozole Drugs 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000000111 anti-oxidant Effects 0.000 description 1
- 230000000890 antigenic Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003886 aromatase inhibitor Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 201000009596 autoimmune hypersensitivity disease Diseases 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid group Chemical group C(C1=CC=CC=C1)(=O)O WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 230000000903 blocking Effects 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 101700018328 ccdB Proteins 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- VYZAMTAEIAYCRO-BJUDXGSMSA-N chromium-51 Chemical compound [51Cr] VYZAMTAEIAYCRO-BJUDXGSMSA-N 0.000 description 1
- 230000002759 chromosomal Effects 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 238000010192 crystallographic characterization Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000004059 degradation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 102000004419 dihydrofolate reductase family Human genes 0.000 description 1
- 108020001096 dihydrofolate reductase family Proteins 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 230000000447 dimerizing Effects 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drugs Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000011051 endospecy test Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- 229960004750 estramustine phosphate Drugs 0.000 description 1
- ADFOJJHRTBFFOF-RBRWEJTLSA-N estramustine phosphate Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)OP(O)(O)=O)[C@@H]4[C@@H]3CCC2=C1 ADFOJJHRTBFFOF-RBRWEJTLSA-N 0.000 description 1
- 239000000328 estrogen antagonist Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229960000255 exemestane Drugs 0.000 description 1
- 230000001747 exhibiting Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000037320 fibronectin Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000000684 flow cytometry Methods 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
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037240 fusion proteins Human genes 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 239000011440 grout Substances 0.000 description 1
- 230000002949 hemolytic Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000003372 histidine group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 230000002209 hydrophobic Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003100 immobilizing Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular Effects 0.000 description 1
- 230000002601 intratumoral Effects 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229960003881 letrozole Drugs 0.000 description 1
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000003340 mental Effects 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000006956 minimum essential medium Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L na2so4 Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000031942 natural killer cell mediated cytotoxicity Effects 0.000 description 1
- 230000001264 neutralization Effects 0.000 description 1
- 230000003472 neutralizing Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003000 nontoxic Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000001575 pathological Effects 0.000 description 1
- WBXPDJSOTKVWSJ-ZDUSSCGKSA-N pemetrexed Chemical compound C=1NC=2NC(N)=NC(=O)C=2C=1CCC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 WBXPDJSOTKVWSJ-ZDUSSCGKSA-N 0.000 description 1
- 229960005079 pemetrexed Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000000546 pharmaceutic aid Substances 0.000 description 1
- 238000011170 pharmaceutical development Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229920002530 poly[4-(4-benzoylphenoxy)phenol] polymer Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002062 proliferating Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 125000001235 proline group Chemical group [H]N1[C@@](C(=O)[*])([H])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000575 proteomic Methods 0.000 description 1
- 230000002285 radioactive Effects 0.000 description 1
- 230000000384 rearing Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 231100000486 side effect Toxicity 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 229960003010 sodium sulfate Drugs 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- WSWCOQWTEOXDQX-UHFFFAOYSA-N sorbic acid Chemical compound CC=CC=CC(O)=O WSWCOQWTEOXDQX-UHFFFAOYSA-N 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 230000003595 spectral Effects 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 229930003347 taxol Natural products 0.000 description 1
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 1
- 229910052713 technetium Inorganic materials 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- LXZZYRPGZAFOLE-UHFFFAOYSA-L transplatin Chemical compound [H][N]([H])([H])[Pt](Cl)(Cl)[N]([H])([H])[H] LXZZYRPGZAFOLE-UHFFFAOYSA-L 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- 239000011778 trisodium citrate Substances 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
- 229960000281 trometamol Drugs 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N β-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—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
- C07K16/2827—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 B7 molecules, e.g. CD80, CD86
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
-
- 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
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
Disclosed is an antibody characterised by having the following properties of: (a) specifically binding to B7-H3; (b) having an antibody-dependent phagocytosis (ADCP) activity; and cell-mediated (c) having an in vivo anti-tumour activity, or a functional fragment of the antibody, wherein said antibody or functional fragment thereof comprises: CDRH1 consisting of an amino acid sequence represented by NYVMH, CDRH2 consisting of an amino acid sequence represented by YINPYNDDVKYNEKFK, and CDRH3 consisting of an amino acid sequence represented by WGYYGSPLYYFDY as complementarity determining regions of the heavy chain; and comprises CDRL1 consisting of an amino acid sequence represented by RASSRLIYMH, CDRL2 consisting of an amino acid sequence represented by ATSNLAS, and CDRL3 consisting of an amino acid sequence represented by QQWNSNPPT as complementarity determining regions of the light chain. Also disclosed is the use of such an antibody for the manufacture of a medicament for treatment of cancer. body or functional fragment thereof comprises: CDRH1 consisting of an amino acid sequence represented by NYVMH, CDRH2 consisting of an amino acid sequence represented by YINPYNDDVKYNEKFK, and CDRH3 consisting of an amino acid sequence represented by WGYYGSPLYYFDY as complementarity determining regions of the heavy chain; and comprises CDRL1 consisting of an amino acid sequence represented by RASSRLIYMH, CDRL2 consisting of an amino acid sequence represented by ATSNLAS, and CDRL3 consisting of an amino acid sequence represented by QQWNSNPPT as complementarity determining regions of the light chain. Also disclosed is the use of such an antibody for the manufacture of a medicament for treatment of cancer.
Description
DESCRIPTION
TITLE OF THE INVENTION: ANTI—B7—H3 ANTIBODY
Technical Field
The present invention relates to an dy which binds
to B7—H3 and.is1iseful.as a'therapeuticzand/or preventive agent
for a tumor, and also s to a method of treating and/or
preventing a tumor using the antibody.
Background Art
B7—H3 is a protein having a single—pass transmembrane
structure (Non—patent document 1). The N—terminal
extracellular domain of B7—H3 contains two variants. Variant
1 contains a V—like and a C—like Ig domain, tively, at
each of two sites, and Variant 2 ns a V~like and a C—like
Ig domain, respectively, at one site. The C—terminal
intracellular domain of B7—H3 contains 45 amino acids.
As a receptor for B7—H3, TLT—Z having a single—pass
transmembrane structure has been reported (Non-patent
document 2). However, there is also a report insisting that
TLT—Z is not a receptor for B7-H3 (Non—patent document 3).
FP1214S l 810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
According to the former report, the activation.of(3D8fipositive
T cells is enhanced when the receptor is bound to B7—H3.
It has been clinically reported that B7—H3 is
pressed in many cancer types, particularly in
non—small—cell lung cancer, kidney cancer, urothelial
oma, colorectal cancer, prostate cancer, glioblastoma
multiforme, ovarian cancer, and1pancreatic cancer (Non—patent
documents 4 to ll). Further, it has been reported that in
prostate cancer, the intensity of sion of B7-H3
positively correlates with clinicopathological malignancy
sudnastumorvolume,extraprostaticinvasion,orGleasonscore,
and also correlates with cancer progression (Non—patent
nt 8). Similarly, in glioblastoma multiforme, the
expression of B7—H3 negatively correlates with event—free
survival (Non—patent document 9), and in pancreatic cancer,
the expression of B7—H3 correlates with lymph node metastasis
and ogical progression (Non—patent document 11). In
ovarian , the expression.of B7~H3 correlates with lymph
node metastasis and pathological progression.
Further, it has been reported that by introducing siRNA
against the B7—H3 gene into a B7~H3—positive cancer cell line,
adhesiveness to fibronectin is reduced thus reducing cell
migration and matrigel invasion (Non—patent document 12).
s 2 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
Furthery it has been reported thatillglioblastomarmiltiforme,
the expression of B7—H3 allows escape from NK cell—mediated
cell death (Non—patent document 13).
Onthecmherhand,B7—H3hasbeenreportedtxnbeexpressed
not only in cancer cells, but also in tumors or surrounding
vessels (Non—patent documents 5&uuil4). It has been reported
that when B7-H3 is sed.in ovarian cancer blood vessels,
the survival rate is sed.
B7 family molecules have been suggested to be d
to the immune system. B7—H3 has been reported.to be expressed
in monocytes, dendritic cells, and ted T cells
(Non—patent document 15). It has been reported that as
cytotoxic: T cells are activated, B7—H3 costimulates the
(proliferation of CD4—positive or CD8—positive T cells.
However, there is also a report that B7—H3 does not play a
costimulatory role (Non-patent document 1).
B7—H3 molecules have been reported to be related to
autoimmune diseases. It has been reported that in rheumatism
and other autoimmune diseases, B7—H3 plays an important role
in the interaction between fibroblast—like synoviocytes and
activated T—cells atent document 16) and that B7—H3
functionsas2acostimulatoryfactorwhencytokinesarereleased
FP1214s 3 WGA/PN810023/3ng trans of PCT 6.9.13
4426167WARENDS
from activated macrophages and ore is related to the
occurrence of sepsis (Non—patent document 17). Further, it
has been reported that by administering an anti—B7—H3 antibody
to a mouse model of asthma during the induction.phase, asthma
is improved due to the suppression of Th2 cell—mediated
cytokine production in regional lymph nodes through the
administration of an anti—mouse B7—H3 antibody (Non—patent
document 18).
With respect to B7—H3, it has been reported that an
antibody against mouse B7—H3 enhances intratumoral
rating CD8-positive1?cells and.suppresses tumor growth
(Non-patent document 14). Further, there is a patent which
discloses that an antibody which izes B7—H3 variant 1
exhibits anzhlviVC>antitumor'effect(xiadenocarcinoma (Patent
document 1).
In spite of these s, an epitope for an anti—B7—H3
antibody which exhibits an in vivo antitumor effect has not
been clarified so far, and there has been no report that a
ic amino acid sequence of the extracellular domain of
B7—H3 is useful as an epitope for a monoclonal antibody for
treating cancer.
Even if antibodies are specific for the same antigen,
the properties of the antibodies may vary due to differences
in epitopes or sequences of the antibodies. Due to the
difference in properties of the dies, when being
clinically administrated to humans, the antibodies exhibit
different reactions in terms of the effectiveness of the
medicinal agent, the frequency of therapeutic response, the
nce of side effects or drug resistance, etc.
Also as regards antibodies against B7—H3, the creation
of an antibody having unprecedented properties has been
strongly demanded.
Related Art Documents
Patent Document
Patent Document 1:
Non—patent Documents
tent Document 1: The Journal of Immunology, 2004,
vol. 172, pp. 2352—2359
tent Document 2: Proceedings of the National
Academy of Sciences of the United States of America, 2008, vol.
105, pp. 10495—10500
tent Document 3: European Journal of Immunology,
2009, vol. 39 pp. l754-l764
FP1214S 5 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
Non—patent Document 4: Lung Cancer, 2009, vol. 66, pp.
245—249
Non—patent Document 5: Clinical Cancer Research, 2008,
vol. 14, pp. 5150—5157
Non—patent Document 6: al Cancer Research 2008,
vol. 14, pp. 4800—4808
Non—patentDocument'h Cancerlmmunology,Immunotherapy,
2010, vol. 59, pp. 1163—1171
Non—patent Document 8: Cancer ch, 2007, vol. 67,
pp. 7893—7900
Non—patent Document 9: Histopathology, 2008, vol. 53,
pp. 73—80
Non—patent Document 10: Modern.Pathology, 2010, vol. 23,
pp. 1104—1112
tent Document 11: British Journal oftiancer, 2009,
vol. 101, pp. 1709—1716
Non—patent Document 12: Current Cancer Drug Targets,
2008, vol. 8, pp. 404~413
Non—patent Document 13: dings of the National
AcademycflfSciencescflftheUnitedStates(IEAmerica,2004,vol.
101, pp. 12640-12645
Non—patent Document.14: Modern Pathology, 2010, vol. 23,
pp. 112
Non—patent Document 15: Nature Immunology, 2001, vol.
2, pp. 269—274
FP1214S 6 WGA/PNBlOOZS/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
Non—patent Document 16: The Journal of logy, 2008 ,
vol. 180, pp. 2989-2998
Non—patent1Document 17: The.Journal.of Immunology; 2010,
vol. 185, pp. 3677—3684
Non—patenILDocument 18: The.Journal.of Immunology; 2008,
vol. 181, pp. 4062—4071
Summary of the ion
ms to be Solved by the Invention
An.object of the invention.is to provide an antibody and
a functional fragment of the antibody' to be used in a
pharmaceutical having a therapeutic effect on a tumor, a method
of treating a tumor using the antibody or a functional fragment
of the antibody, and the like, or to at least provide the public
with a useful choice.
Means for Solving the Problems
The present inventors made intensive studies in order
to achieve the above object, and as a , they found an
antibody which specifically binds to B7—H3 to exhibit an
mor activity, and thus completed the invention. That
is, the invention includes the following inventions.
(1) An antibody characterized by having the following
properties of:
(a) specifically binding to B7—H3;
(b) having an antibody—dependent cell—mediated
phagocytosis (ADCP) activity; and
(c) having an in vivo antitumor ty,
or a functional fragment of the antibody.
(2) The antibody or a functional nt of the antibody
according to the above (1), wherein B7—H3 is a leecule
including an amino acid sequence represented by SEQ ID NO: 6
or 10.
(3)Theantibodyoreafunctionalfragmentoftheantibody
according to the above (1) or (2), which binds to IgCl and/or
IgC2 each of which is a domain of B7—H3.
(4)Theantibodyoraafunctionalfragmentoftheantibody
according to the above (3), wherein IgCl is a<domain including
an amino acid ce represented by amino acid numbers 140
to 244 in SEQ ID NO: 6, and IgC2 is a domain including an amino
acid ce represented by amino acid numbers 358 to 456 in
SEQ ID NO: 6.
(5) The antibodycnta functional fragment.oftjuaantibody
according to any one of the above (1) to (4), which has a
competitive inhibitory activity against M30 antibody for the
binding to B7—H3.
(6)Theantibodyorwafunctionalfragmentoftheantibody
according to any one of the above (1) to (5), which has an
FP1214s 8 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
antibody—dependent cellular cytotoxicity (ADCC) ty
and/or a complement—dependent cytotoxicity (CDC) activity.
(7) The antibody or a functional nt of the antibody
according to any one of the above (1) to (6) , wherein the tumor
is cancer.
(8) The dy or a functional fragment of the antibody
according to the above (7) , wherein the cancer is lung cancer,
breast cancer, te cancer, pancreatic cancer, ctal
cancer, a melanoma, liver cancer, n cancer, bladder
cancer, stomach cancer, esophageal cancer, or kidney cancer.
(9) The antibody or a functional fragment of the antibody
according to any one of the above (1) to (8) , which comprises
CDRHl consisting of an amino acid sequence represented by SEQ
IE NO: 92, CDRHZ consisting of an amino acid sequence
represented by SEQ ID NO: 93, and CDRH3 consisting of an amino
acid sequence represented by SEQ ID NO: 94 as complementarity
determining regions of the heavy chain and comprises CDRLl
consisting of an amino acid sequence represented by SEQ ID NO:
95, CDRL2 consisting of an amino acid sequence represented by
SEQ ID NO: 96, and CDRL3 consisting of an amino acid sequence
represented by SEQ ID NO: 97 as complementarity determining
regions of the light chain.
(10) The antibody or a functional fragment of the
antibody according to any one of the above (1) to (9) , which
comprises a heavy chain variable region consisting of an amino
FP12145 9 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
acid sequence represented by amino acid numbers 20 to 141 in
SEQ ID NO: 51 and a light chain variable region consisting of
an amino acid sequence represented by amino acid numbers 23
to 130 in SEQ ID NO: 53.
(11) The antibody or a functional fragment of the
antibody'accordingtx>any'one of the above (1) to (10), wherein
a constant region is a human—derived constant region.
(12) The dy or a functional fragment of the
dy according to the above (11), which comprises a heavy
chain consisting of an amino acid sequence represented by SEQ
ID NO: 63 and a light chain consisting of an amino acid ce
represented by SEQ ID NO: 59.
(13) The antibody or a functional fragment of the
antibody according to any one of the above (1) to (12), which
is humanized.
(14) The antibody or 51 functional fragment of the
antibody ing to the above (13), which comprises: a heavy
chain variable region consisting of an amino acid sequence
edfromthegroupconsistingof HM anaminoacidsequence
represented.by amino acid numbers 20 to 141 in SEQ ID NO: 85,
(b) an amino acid sequence represented by amino acid numbers
20tol41inSEQIDIMk 87,(c)anaminoacidsequencerepresented
by amino acid numbers 20 to 141 in SEQ ID NO: 89, (d) an amino
acid sequence ented by amino acid numbers 20 to 141 in
FP12l4s 10 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
SEQ ID NO: 91, (e) an amino acid sequence having a gy
of at least 95% or more with any of the sequences (a) to (d),
and (f) an amino acid sequence wherein one or several amino
acids are deleted, substituted or added in any of the sequences
(a) to (d); and a light chain le region consisting of
an amino acid sequence ed from the group consisting of
(g) an amino acid sequence represented by amino acid numbers
21tol28:U1SEQIDIMh 71,(h)anaminoacidsequencerepresented
by amino acid numbers 21 to 128 in SEQ ID NO: 73, (i) an amino
acid sequence represented by amino acid numbers 21 to 128 in
SEQ ID NO: 75, (j) an amino acid sequence represented by amino
acid numbers 21 to 128 in SEQ ID NO: 77, (k) an amino acid
sequence represented by amino acid s 21 to 128 in SEQ
I[)NO: 79, (1) an amino acid.sequence represented by amino acid
numbers 21 to 128 in SEQ ID NO: 81, (m) an amino acid sequence
represented by amino acid numbers 21 to 128 in SEQ ID NO: 83,
(n) an amino acid sequence having a homology of at least 95%
or more with any of the sequences (g) to (m), and (0) an amino
acid sequence wherein one or several amino acids are deleted,
substituted or added in any of the sequences (g) to (m).
(15) The antibody or a functional fragment of the
antibody according to the above (14), which comprises a heavy
chain variable region and a light chain variable region
selected from the group consisting of: a heavy chain variable
region consisting of an amino acid sequence ented by
FP1214s 11 WGA/PN8lOOZ3/Eng trans of PCT spec/26.9.13
4426167WARENDS
amino acid s 20 to 141 in SEQ ID NO: 85 and a light chain
variable region consisting of an amino acid sequence
represented by amino acid numbers 21 to 128 in SEQ ID NO: 71;
a heavy chain variable region consisting of an amino acid
sequence represented by amino acid numbers 20 to 141 in SEQ
ID NO: 85 and a light chain variable region consisting of an
amino acid sequence represented by amino acid numbers 21 to
128 in SEQ ID NO: 73; a heavy chain variable region consisting
of an amino acid sequence ented by amino acid numbers
to 141 in SEQ ID NO: 85 and a light chain variable region
consisting ofzniamino acid sequence representedknzamino acid
numbers 21 to 128 in SEQ ID NO: 75; a heavy chain variable region
ting<xfan,amino acid.sequence representedhmramino acid
numbers 20 to 141 in SEQ ID NO: 85 and a light chain variable
region consisting of an amino acid sequence represented by
amino acid numbers 21 to 128 in SEQ ID NO: 77; a heavy chain
variable region consisting of an amino acid sequence
represented by amino acid numbers 20 to 141 in SEQ ID NO: 85
and a light chain le region consisting of an amino acid
sequence represented by amino acid numbers 21 to 128 in SEQ
II)NO: 79; a heavy chain variable region.consisting of an amino
acid sequence ented by amino acid numbers 20 to 141 in
SEQ ID NO: 85 and a light chain variable region consisting of
an amino acid ce represented by amino acid numbers 21
t0128:hiSEQ]IDNO:81;aaheavychainvariableregionconsisting
FP1214s l2 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
of an amino acid sequence represented by amino acid numbers
to 141 in SEQ ID NO: 85 and a light chain variable region
consisting ofan amino acid sequence represented.by amino acid
numbers 21 to 128 in SEQ ID NO: 83; a heavy chain variable region
consisting of an amino acid sequence representedknzamino acid
numbers 20 to 141 in SEQ ID NO: 91 and a light chain variable
region consisting of an amino acid sequence ented by
amino acid numbers 21 to 128 in SEQ ID NO: 71; a heavy chain
variable region ting of an amino acid sequence
represented by amino acid numbers 20 to 141 in SEQ ID NO: 91
and a light chain variable region consisting of an amino acid
sequence ented by amino acid numbers 21 to 128 in SEQ
I£)NO: 73; a heavy chain variable region consisting of an amino
acid sequence represented by amino acid numbers 20 to 141 in
SEQ ID NO: 91 and a light chain variable region consisting of
an amino acid sequence represented by amino acid numbers 21
to 128 in SEQ ID NO: 75; and a heavy chain le region
consisting of an amino acid sequence representedknzamino acid
numbers 20 to 141 in SEQ ID NO: 91 and a light chain variable
region consisting of an amino acid sequence ented by
amino acid numbers 21 to 128 in SEQ ID NO: 77.
(16) The antibody or a functional fragment of the
antibody according to the above (14) or (15), which comprises
a heavy chain and a light chain selected from the group
consisting of: a heavy chain consisting of an amino acid
FP1214s 13 810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
sequence represented by amino acid numbers 20 to 471 in SEQ
ID NO: 85 and a light chain consisting of an amino acid sequence
represented.by amino acid numbers 21 to 233 in SEQ ID NO: 71;
a heavy chain consisting of an amino acid sequence represented
by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light
chainconsistingofeniaminoacidsequencerepresenteibyamino
acidnumberleix>233inSEQZUDNO:73;aiheavychainconsisting
of an amino acid sequence ented by amino acid numbers
to 471 in SEQ ID NO: 85 and a light chain consisting of an
amino acid sequence represented by amino acid s 21 to
233 in SEQ ID NO: 75; a heavy chain consisting of an amino acid
sequence represented by amino acid numbers 20 to 471 in SEQ
ID NO: 85 and a light chain ting of an amino acid sequence
represented.by amino acid numbers 21 to 233 in SEQ ID NO: 77;
a heavy chain consisting of an.amino acid sequence represented
by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light
chainconsistingofaniaminoacidsequencerepresentaibyamino
acidnumberletx3233inSEQZUDNO:79;aaheavychainconsisting
of an amino acid sequence ented by amino acid numbers
to 471 in SEQ ID NO: 85 and a light chain consisting of an
amino acid sequence represented by amino acid numbers 21 to
233 in SEQ ID NO: 81; a heavy chain consisting of an amino acid
sequence represented by amino acid numbers 20 to 471 in SEQ
ID NO: 85 and a light chain consisting of an amino acid sequence
represented.by amino acid numbers 21 to 233 in SEQ ID NO: 83;
E‘P1214s 14 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
a heavy chain consisting of an amino acid sequence represented
by amino acid numbers 20 to 471 in SEQ ID NO: 91 and a light
chainconsistingofaniaminoacidsequencerepresentedbyamino
acidnumberletx>233inSEQIUDNO:71;aiheavychainconsisting
of an amino acid sequence represented by amino acid numbers
to 471 in SEQ ID NO: 91 and a light chain consisting of an
amino acid sequence represented by amino acid numbers 21 to
233 in SEQ ID NO: 73; a heavy chain consisting of an amino acid
sequence represented by amino acid numbers 20 to 471 in SEQ
IDIKh 91andaalightchainconsistingofaniaminoacidsequence
represented by amino acid numbers 21 to 233 in SEQ ID NO: 75;
and a heavy chain consisting of an amino acid sequence
ented by amino acid numbers 20 to 471 in SEQ ID NO: 91
and. a light chain consisting of an amino acid sequence
represented by amino acid s 21 to 233 in SEQ ID NO: 77.
(17) The antibody or a onal fragment of the
antibody according to any one of the above (14) to (16), which
comprises a heavy chain and a light chain selected from the
group consisting of: a heavy chain consisting of an amino acid
sequence represented by SEQ ID NO: 85 and a light chain
consisting of an amino acid sequence ented.by SEQ ID NO:
71; a heavy chain consisting of an amino acid sequence
represented by SEQ ID NO: 85 and a light chain consisting of
an amino acid sequence ented by SEQ ID NO: 73; a heavy
chain consisting of an amino acid sequence represented by SEQ
FP1214S 15 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
ID NO: 85 and a light chain consisting of an amino acid sequence
represented by SEQ ID NO: 75; a heavy chain consisting of an
amino acid sequence represented by SEQ ID NO: 85 and a light
chain consisting of an amino acid ce ented by SEQ
ID NO: 77; a heavy chain consisting of an amino acid sequence
represented by SEQ ID NO: 85 and a light chain consisting of
an amino acid sequence represented by SEQ ID NO: 79; a heavy
chain consisting of an amino acid sequence ented by SEQ
ID NO: 85 and a light chain consisting of an amino acid sequence
represented by SEQ ID NO: 81; a heavy chain consisting of an
amino acid sequence ented by SEQ ID NO: 85 and a light
chain consisting of an amino acid sequence represented by SEQ
ID NO: 83; a heavy chain consisting of an amino acid sequence
represented by SEQ ID NO: 91 and a light chain consisting of
an amino acid sequence represented by SEQ ID NO: 71; a heavy
chain ting of an amino acid sequence represented by SEQ
ID NO: 91 and a light chain consisting of an amino acid sequence
represented by SEQ ID NO: 73; a heavy chain ting of an
amino acid sequence represented by SEQ ID NO: 91 and a light
chain consisting of an amino acid sequence represented by SEQ
IDIMR 75;andeaheavychainconsistingofamiaminoacidsequence
represented by SEQ ID NO: 91 and a light chain consisting of
an amino acid sequence represented by SEQ ID NO: 77.
(18) The functional fragment of the antibody according
to any one of the above (1) to (17), wherein the functional
FP1214S 16 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
fragment is selected.fron1the group consisting of Fab, F(ab)2,
Fab’ and Fv.
(19) A polynucleotide encoding the antibody or a
functional nt of the antibody according to any one of
the above (1) to (18).
(20) The polynucleotide according to the above (19),
which comprises a nucleotide sequence represented by
nucleotide numbers 581x3423 in SEQZHDNO: a nucleotide
sequence represented by nucleotide numbers 67 to 390 in SEQ
ID NO: 52.
(21) The polynucleotide according to the above (19) or
(20), which includes a nucleotide sequence represented.by SEQ
ID NO: 62 and a nucleotide sequence represented by SEQ ID NO:
(22) The polynucleotide ing to the above (19) or
(20), which comprises: a.nucleotide sequence selected.fron1the
group consisting of (a) a nucleotide sequence represented by
nucleotide numbers 58 to 423 in SEQ IDDKh 84, (b)2anucleotide
sequence represented by nucleotide numbers 58 to 423 in SEQ
IDDKM 86, kfl a nucleotide sequence entedknznucleotide
s 58 to 423 in SEQ ID NO: 88, (d) a nucleotide sequence
represented.by tide numbers 58 to 423 in SEQ ID NO: 90,
and «fl anucleotidesequencecomprisingaapolynucleotidewhich
hybridizes to a polynucleotide ting of a nucleotide
sequence complementary to any of the nucleotide sequences (a)
FP1214s l7 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
to (d) under stringent conditions; and a nucleotide sequence
selected from the group consisting of (f) a nucleotide sequence
represented by nucleotide numbers 61 to 384 in SEQ ID NO: 70,
(g) a nucleotide sequence represented by tide numbers
61to384:U1SEQIDNO:72, MU otidesequencerepresented
bynucleotidenumbers61113384inSEQZUDNO:74, UJ anucleotide
sequence ented by nucleotide numbers 61 to 384 in SEQ
II)NO: 76,(j) a nucleotide sequence representedkuznucleotide
numbers 61 to 384 in SEQ ID NO: 78, (k) a nucleotide sequence
represented.by nucleotide numbers 61 to 384 in SEQ ID NO: 80,
(1) a nucleotide sequence represented by nucleotide numbers
61 to 384 in SEQ ID NO: 82, and (m) a nucleotide sequence
comprising a polynucleotide which hybridizes to a
cleotide consisting of a nucleotide sequence
complementary to any of the nucleotide sequences (f) to (1)
under stringent conditions.
(23) The polynucleotide according to the above (22),
which comprises nucleotide sequences selected from the group
tingof:aanucleotidesequencerepresentedbyrumfleotide
numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide sequence
represented by nucleotide numbers 61 to 384 in SEQ ID NO: 70;
a nucleotide sequence represented by nucleotide numbers 58 to
423 in SEQ ID NO: 84 and a tide sequence represented by
tide numbers 61 to 384 in SEQ ID NO: 72; a nucleotide
sequence represented by nucleotide numbers 58 to 423 in SEQ
FP1214S 18 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
ID NO: 84 and a nucleotide sequence represented by nucleotide
numbers 61 to 384 in SEQ ID NO: 74; a tide sequence
represented by nucleotide numbers 58 to 423 in SEQ ID NO: 84
and a nucleotide sequence represented by nucleotide numbers
61 to 384 in SEQ ID NO: 76; a nucleotide sequence represented
bynucleotidenumbers58'U3423jleEQIDIMk 84andaanucleotide
sequence represented by nucleotide numbers 61 to 384 in SEQ
ID NO: 78; a nucleotide sequence represented by nucleotide
numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide sequence
represented by nucleotide numbers 61 to 384 in SEQ ID NO: 80;
a nucleotide sequence represented by nucleotide numbers 58 to
423 in SEQ ID NO: 84 and a nucleotide sequence represented by
nucleotide numbers 61 to 384 in SEQ ID NO: 82; a nucleotide
sequence represented by nucleotide numbers 58 to 423 in SEQ
ID NO: 90 and a tide ce represented by nucleotide
s 61 to 384 in SEQ ID NO: 70; a nucleotide sequence
represented by nucleotide numbers 58 to 423 in SEQ ID NO: 90
and a nucleotide sequence represented by nucleotide numbers
61 to 384 in SEQ ID NO: 72; a nucleotide ce represented
eotidenumbers58 U3423i118EQIDIKh 90and¢anucleotide
sequence ented by nucleotide numbers 61 to 384 in SEQ
IDDKX 74; andaanucleotide sequence representequrnucleotide
numbers 58 to 423 in SEQ ID NO: 90 and a nucleotide sequence
represented by tide numbers 61 to 384 in SEQ ID NO: 76.
(24) The polynucleotide according to the above (22) or
FP1214s 19 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
(23), which comprises nucleotide sequences selected from the
group consisting of: a nucleotide sequence represented by
nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide
sequence represented by nucleotide numbers 61 to 699 in SEQ
ID NO: 70; a tide sequence represented by nucleotide
s 58 to 1413 in SEQ ID NO: 84 and a nucleotide ce
represented by nucleotide numbers 61 to 699 in SEQ ID NO: 72;
a nucleotide sequence represented by nucleotide numbers 58 to
1413 in SEQ ID NO: 84 and a tide sequence represented
by nucleotide numbers 61 to 699 in SEQ ID NO: 74; a nucleotide
sequence represented by nucleotide numbers 58 to 1413 in SEQ
ID NO: 84 and a nucleotide sequence represented by nucleotide
numbers 61 to 699 in SEQ ID NO: 76; a nucleotide sequence
represented by nucleotide s 58 to 1413 in SEQ ID NO: 84
and a nucleotide sequence ented by nucleotide numbers
61 to 699 in SEQ ID NO: 78; a nucleotide sequence represented
bynucleotidenumbers58tol413inSEQIDth 84andarnmfleotide
sequence represented by tide numbers 61 to 699 in SEQ
ID NO: 80; a nucleotide sequence represented by nucleotide
numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide sequence
represented by nucleotide numbers 61 to 699 in SEQ ID NO: 82;
a nucleotide sequence represented by nucleotide numbers 58 to
1413 in SEQ ID NO: 90 and a nucleotide sequence ented
by nucleotide numbers 61 to 699 in SEQ ID NO: 70; a nucleotide
sequence represented.by nucleotide numbers 58 to 1413 in SEQ
FP1214s 2O WGA/PN810023/Eng trans of PCT 6.9.13
4426157—1—WARENDS
ID NO: 90 and a nucleotide ce represented by nucleotide
numbers 6l to 699 in SEQ ID NO: 72; a nucleotide sequence
represented by nucleotide numbers 58 to 1413 in SEQ ID NO: 90
and a nucleotide sequence represented by nucleotide numbers
61 to 699 in SEQ ID NO: 74; and a nucleotide sequence represented
by nucleotide numbers 58 to 1413 in SEQ ID NO: 90 and a nucleotide
sequence represented by tide numbers 61 to 699 in SEQ
ID NO: 76.
(25) The polynucleotide according to any one of the above
(22) to (24), which comprises nucleotide sequences selected
from the group consisting of: a nucleotide sequence ented
by SEQ ID NO: 84 and a nucleotide sequence represented by SEQ
ID NO: 70; a nucleotide sequence represented by SEQ ID NO: 84
and a nucleotide ce represented by SEQ ID NO: 72; a
nucleotide sequence represented by SEQ ID NO: 84 and a
nucleotide sequence represented by SEQ ID NO: 74; a nucleotide
sequence represented by SEQ ID NO: 84 and a nucleotide sequence
represented by SEQ ID NO: 76; a nucleotide sequence represented
by SEQ ID NO: 84 and a nucleotide sequence represented by SEQ
ID NO: 78; a nucleotide sequence represented by SEQ ID NO: 84
and a nucleotide sequence represented by SEQ ID NO: 80; a
nucleotide sequence represented by SEQ ID NO: 84 and a
nucleotide ce represented by SEQ ID NO: 82; a nucleotide
sequence represented by SEQ ID NO: 90 and a nucleotide sequence
represented by SEQ ID NO: 70; a nucleotide ce represented
21 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
by SEQ ID NO: 90 and a nucleotide sequence represented by SEQ
ID NO: 72; a nucleotide sequence represented by SEQ ID NO: 90
and a nucleotide sequence represented by SEQ ID NO: 74; and
a nucleotide sequence represented by SEQ ID NO: 90 and a
nucleotide sequence ented by SEQ ID NO: 76.
(26) An expression vector including a polynucleotide
according to any one of the above (19) to (25).
(27) A host cell, which is transformed. with the
expression vector according to the above (26).
(28) The host cell according to the above (27), wherein
the host cell is a eukaryotic cell.
(29) A method of producing an antibody or a functional
fragment of the dy, characterized by ing a step
of culturing the host cell according to the above (27) or (28)
and a step of collecting a desired antibody or a functional
fragment of the antibody from a cultured product obtained in
the culturing step.
(30) An antibody or a functional fragment of the antibody,
characterized by being obtained by the production method
according to the above (29).
(31) The functional fragment of the dy according
to the above (30), wherein the functional fragment is selected
from the group ting of Fab, F(ab)2, Fab’ and Fv.
(32) The antibody or 23 functional fragment of the
FP1214s 22 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
antibody according to any one of the above (1) to (18), (30),
and (31), wherein the modification of a glycan is regulated
tc>enhanceen1antibody-dependent cellular xic1activity.
(33) A pharmaceutical composition terized by
ing' at least one of the antibodies or functional
fragments of the antibodies according to the above (1) to (18),
and (30) to (32).
(34) The pharmaceutical composition according to the
above (33), which is for treating a tumor.
(35) A pharmaceutical composition for treating a tumor
characterized by including at least one of the antibodies or
functional fragments of the dies according to the above
(1) to (18), and (30) to (32) and.at least one therapeutic agent
for cancer.
(36) The pharmaceutical composition according to the
above (34) or (35), wherein the tumor is cancer.
(37) The pharmaceutical composition according to the
above (36), wherein the cancer is lung , breast cancer,
prostate cancer, pancreatic cancer, colorectal cancer, a
melanoma, liver cancer, ovarian cancer, bladder cancer,
stomach , esophageal cancer, or kidney cancer.
(38) A method of treating a tumor, characterized by
administering at least one of the antibodies or functional
s 23 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
fragments(fiftheantibodiesaccordingixntheabove HJ to(l8),
and (30) to (32) to an individual.
(39) A method of treating a tumor, characterized by
administering at least one of the antibodies or functional
fragmentscflftheantibodiesaccordingtxntheabove HJ to(l8),
and (30) to (32) and.at least one therapeutic agent for cancer
simultaneously, separately, or sequentiallyiuaan individual.
(40) The treatment method ing to the above (38)
or (39), wherein the tumor is cancer.
(41) The treatment method according to the above (40),
wherein the cancer is lung cancer, breast cancer, prostate
cancer, pancreatic cancer, colorectal , a melanoma,
liver cancer, ovarian cancer, bladder cancer, stomach cancer,
esophageal , or kidney cancer.
Advantage of the Invention
According to the invention, a therapeutic agent or the
like for cancer comprising an dy which binds to B7—H3
and has an antitumor activity against cancer cells can be
obtained.
Brief Description of the Drawings
FP1214S 24 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
[Fig. 1] Fig. l is a View showing the presence or absence
of ADCP activity of an anti—B7—H3 antibody t NCI-H322
cells. Theerrorbarslethedrawingrepresentstandarderrors
(n=3).
[Fig. 2] Figu 2 is a view'showing the presence or absence
of ADCP activity’ of a commercially' available anti—B7—H3
antibody against NCI—H322 cells.
[Fig. 3] Fig. 3 is a view showing the presence or absence
of ADCC activity of M30 antibody against empty
vector—transfected 293 cells and B7—H3—expressing 293 cells.
The error bars in the drawing represent standard errors (n=3).
In the drawing, the “mock” s the ADCC activity of M30
against empty vector—transfected 293 cells, and the “B7H3”
denotes the ADCC activity of M30 against B7—H3—expressing 293
cells.
[Fig. 4] Fig; 4 is a View showing the presence or absence
of CDC activity of an anti—B7—H3 dy against NCI—H322
cells. orbarsixlthedrawingrepresentstandarderrors
(n=3).
[Fig. 5—1] Fig. 5~l is a view showing the reactivity of
M30 antibody against a B7—H3 ent mutant (B7—H3 Ing).
The dotted line indicates the binding property of a control
antibody, and the solid line indicates the binding property
of an M30 antibody.
[Fig. 5—2] Fig. 5—2 is a view g the reactivity of
M30 antibody against a B7—H3 deficient mutant (B7-H3 IgCl).
The dotted line indicates the binding property of a control
antibody, and the solid line indicates the binding property
of an M30 dy.
[Fig. 5—3] Fig. 5—3 is a view showing the reactivity of
M30 antibody against a B7—H3 deficient mutant (B7—H3 IgV2).
The dotted line indicates the binding property of a l
antibody, and the solid line indicates the binding property
of an M30 antibody.
[Fig. 5—4] Fig. 5—4 is a view g the reactivity of
M30 dy against a B7—H3 deficient mutant (B7—H3 IgC2).
The dotted line tes the binding property of a control
antibody, and the solid line indicates the binding property
of an M30 antibody.
[Fig. 5—5] Fig. 5—5 is a view showing the reactivity of
M30 antibody against a B7—H3 deficient mutant (B7—H3
IgCl-VZ—CZ). The dotted line indicates the binding property
of a control antibody, and the solid line tes the binding
property of an M30 antibody.
[Fig. 5—6] Fig. 5—6 is a view showing the reactivity of
M30 dy'againstaaB7—H3 deficientlnutant (B7—H3 IgV2-C2).
The dotted line indicates the binding property of a control
antibody, and the solid line indicates the binding property
of an M30 antibody.
[Fig. 6] Fig. 6 is a View showing the antitumor activities
of an anti—B7—H3 antibodies against mice implanted with
22 cells. The error bars in the drawing represent
rd errors (n=lO).
[Fig. 7] Fig; 7 is a view showing the antitumor ty
of M30 dy when macrophages were depleted in vivo. The
error bars in the drawing ent standard errors (n=8).
Further, the “mm“3” denotes \\ 3 //
[Fig. 8] Fig. 8 is a view showing the ADCP activities
of M30 antibody and.cM30 antibody against NCI—H322 cells. The
error bars in the drawing represent standard errors (n=4).
[Fig.SH Fig.9i1;aviewshowingtheantitumoractivities
of M30 antibody and cM30 dy against mice implanted with
MDA-MB—23l cells. The error bars in the drawing represent
standard errors (n=9).
[Fig. 10—1] Fig. 10—1 is a view showing the competitive
inhibitory activities of cM30 antibody'andIH30—Hl—L4 antibody
against M30 for the binding to an extracellular domain
polypeptide antigen of a B7—H3 variant 1 antigen. The error
bars in the drawing represent standard errors (n=3).
[Fig. 10—2] Fig. 10—2 is a view showing the competitive
inhibitory activities of cM3O antibody and1M30—Hl—L4 antibody
against M30 for the binding' to an extracellular domain
polypeptide antigen of a B7—H3 variant 2 antigen. The error
bars in the drawing represent standard errors (n=3).
[Fig. 11] Fig. ll is a view g the ADCP activities
FPlZl4s 2'7 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
ofMBOantibody,cMBOantibody,andNBO—Hl—L4antibodyagainst
NCI—H322 cells. The error bars in the drawing ent
standard errors (n=4).
[Fig. 12] Fig. 12 is a view showing the ADCC activities
ofcM30antibodyandMBO—Hl—L4antibodyagainstNCI—H322cells.
The error bars in the drawing represent standarrierrors (n=3).
[Fig. 13—1] Fig. 13—1 shows a nucleotide sequence of
B7—H3 variant 1 (SEQ ID NO: 5).
[Fig. 13-2] Fig. 13-2 shows an amino acid sequence of
the B7-H3 variant 1 (SEQ ID NO: 6).
[Fig. 14—1] Fig. 14—1 shows a nucleotide ce of
B7—H3 variant 2 (SEQ ID NO: 9).
[Fig. 14—2] Fig. 14—2 shows an amino acid sequence of
the B7—H3 variant 2 (SEQ ID NO: 10).
[Fig. 15—1] Fig. 15-1 shows a nucleotide sequence of
B7-H3 Ing (SEQ ID NO: 20).
[Fig. 15—2] Fig. 15—2 shows an amino acid sequence of
the B7~H3 Ing (SEQ ID NO: 21).
[Fig. 16—1] Fig. 16—1 shows a nucleotide sequence of
B7-H3 IgCl (SEQ ID NO: 22) .
[Fig. 16—2] Fig. 16—2 shows an amino acid sequence of
the B7—H3 IgCl (SEQ ID NO: 23).
[Fig. 17—1] Fig. 17—1 shows a nucleotide sequence of
B7—H3 IgV2 (SEQ ID NO: 24).
[Fig. 17—2] Fig. 17—2 shows an amino acid ce of
FP1214s 253 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
the B7—H3 IgV2 (SEQ ID NO: 25).
[Fig. 18—1] Fig. 18—1 shows a nucleotide ce of
B7—H3 IgC2 (SEQ ID NO: 26).
[Fig. 18—2] Fig. 18—2 shows an amino acid sequence of
the B7-H3 IgC2 (SEQ ID NO: 27) .
[Fig. 19—1] Fig. 19-1 shows a nucleotide sequence of
B7-H3 IgCl-VZ-CZ (SEQ ID NO: 28).
[Fig. 19—2] Fig. 19-2 shows an amino acid sequence of
the B7—H3 IgCI—VZ—C2 (SEQ ID NO: 29).
[Fig. 20—1] Fig. 20—1 shows a nucleotide sequence of
B7—H3 IgV2—C2 (SEQ ID NO: 30).
[Fig. 20—2] Fig. 20—2 shows an amino acid sequence of
B7-H3 IgV2-C2 (SEQ ID NO: 31).
[Fig. 21-1] Fig. 21—1 shows a nucleotide sequence of an
M30 antibody heavy chain (SEQ ID NO: 50).
[Fig. 21—2] Fig. 21-2 shows an amino acid sequence of
the M30 antibody heavy chain (SEQ ID NO: 51).
[Fig. 22—1] Fig. 22-1 shows a nucleotide sequence of an
M30 antibody light chain (SEQ ID NO: 52).
[Fig. 22—2] Fig. 22-2 shows an amino acid sequence of
the M30 antibody light chain (SEQ ID NO: 53).
[Fig. 23] Fig. 23 shows a nucleotide ce of a human
K chain secretory signal” a humanlcchain constant region, and
a human poly-A onal signal (SEQ ID NO: 56).
[Fig. 24] Fig. 24 shows armicleotide sequence of a:signal
FPlZlés 29 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
sequence and a constant region of human IgG1 (SEQ ID 57).
[Fig. 25—1] Fig. 25—1 shows a nucleotide sequence of an
M30 antibody chimera—type light chain (SEQ ID NO: 58).
[Fig. 25—2] Fig. 25-2 shows an amino acid ce of
the M30 antibody chimera—type light chain (SEQ ID NO: 59).
[Fig. 26—1] Fig. 26—1 shows a nucleotide sequence of an
M30 antibody chimera-type heavy chain (SEQ ID NO: 62).
[Fig. 26—2] Fig. 26—2 shows an amino acid sequence of
the M30 antibody chimera—type heavy chain (SEQ ID NO: 63).
[Fig. 27—1] Fig. 27—1 shows a nucleotide sequence of an
MBO—Ll—type light chain (SEQ ID NO: 70).
[Fig. 27—2] Fig. 27—2 shows an amino acid sequence of
the M30-L1—type light chain (SEQ ID NO: 71).
[Fig. 28—1] Fig. 28—1 shows a nucleotide sequence of an
—type light chain (SEQ ID NO: 72).
[Fig. 28—2] Fig. 28—2 shows an amino acid sequence of
the M30—L2—type light chain (SEQ ID NO: 73).
[Fig. 29-1] Fig. 29—1 shows a nucleotide ce of an
M30—L3—type light chain (SEQ ID NO: 74).
[Fig. 29—2] Fig. 29—2 shows an amino acid sequence of
the M30-L3-type light chain (SEQ ID NO: 75).
[Fig. 30—1] Fig. 30—1 shows a nucleotide sequence of an
M30—L4—type light chain (SEQ ID NO: 76).
[Fig. 30-21 Fig. 30—2 shows an amino acid ce of
the M30—L4—type light chain (SEQ ID NO: 77).
FP1214s 3O WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
[Fig. 31—1] Fig. 31—1 shows a nucleotide ce of an
M30—L5—type light chain (SEQ ID NO: 78).
[Fig. 31—2] Fig. 31-2 shows an amino acid sequence of
the M30—L5—type light chain (SEQ ID NO: 79).
[Fig. 32—1] Fig. 32—1 shows a nucleotide sequence of an
M30—L6—type light chain (SEQ ID NO: 80).
[Fig. 32-2] Fig. 32—2 shows an amino acid sequence of
the M30-L6-type light chain (SEQ ID NO: 81).
[Fig. 33—1] Fig. 33—1 shows a nucleotide ce of an
M30-L7—type light chain (SEQ ID NO: 82).
[Fig. 33—2] Fig. 33—2 shows an amino acid sequence of
the M30—L7-type light chain (SEQ ID NO: 83).
[Fig. 34—1] Fig. 34—1 shows a nucleotide sequence of an
MBO—Hl—type heavy chain (SEQ ID NO: 84).
[Fig. 34—2] Fig. 34—2 shows an amino acid sequence of
the MBO—Hl—type heavy chain (SEQ ID NO: 85).
[Fig. 35—1] Fig. 35-1 shows a nucleotide sequence of an
—type heavy chain (SEQ ID NO: 86).
[Fig. 35—2] Fig. 35-2 shows an amino acid sequence of
the M30—H2—type heavy chain (SEQ ID NO: 87).
[Fig. 36—1] Fig. 36—1 shows a nucleotide sequence of an
M30—H3—type heavy chain (SEQ ID NO: 88).
[Fig. 36-2] Fig. 36—2 shows an amino acid sequence of
the M30-H3-type heavy chain (SEQ ID NO: 89).
[Fig. 37—1] Fig. 37—1 shows a nucleotide sequence of an
FP1214s 31 WGA/PNBIOOZB/Eng trans of PCT spec/26.9.13
4426167WARENDS
M30—H4—type heavy chain (SEQ ID NO: 90).
[Fig. 37—2] Fig. 37—2 shows an amino acid sequence of
the M30—H4—type heavy chain (SEQ ID NO: 91).
8]Fig.38i1;aviewshowingtheantitumoractivity
of a humanized M30 (M30—Hl—L4) antibody against mice implanted
withMDA—MB—231cells. Theerrorbarsinthedrawingrepresent
standard errors (n=6).
Mode for Carrying Out the Invention
The terms r” and “tumor” as used herein are used
with the same meaning.
The term “gene” as used herein includes not only DNA,
but also mRNA thereof, cDNA thereof and cRNA thereof.
The term “polynucleotide” as used herein is used with
the same meaning as a nucleic acid and also includes DNA, RNA,
probes, oligonucleotides, and primers.
The terms “polypeptide” and.“proteiJV’as used herein.are
used t distinction.
The term “cell” as used herein also includes cells in
an animal individual and cultured cells.
FP1214S 32 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
The term “B7—H3” as used herein is used in the same
g as B7—H3 protein, and also refers to B7—H3 variant 1
and/or B7—H3 t 2.
The term “cell injury” as used herein refers to a state
ir1whicheapathological.changejjscausedix>cellsillsome form,
andthecellinjuryisrmmflufldtedtochrectinjuryandincludes
all sorts ofcdamage to the structure and.functicmlof cells such
as DNA cleavage, base-dimer ion, chromosomal cleavage,
damage to cell division machinery, and a decrease in various
enzymatic activities.
The term “cytotoxic activity” as used herein refers to
the activity of causing the above—described cell injury.
The term. “antibody-dependent ediated
phagocytosis activity” as used herein refers to
“antibody—dependent cell phagocytosis (ADCP) activity” and
means the activitycfifphagocytosing target cells such as tumor
cellsbynwnocytesornacrophagesnediatedknzanantibody. The
term is also called “antibody—dependent phagocytosis
activity”.
The term. “antibody—dependent cellular cytotoxicity
activity” as used herein refers to “antibody—dependent
cellular cytotoxicity (ADCC) activity” andIneans the activity
of ng target cells such as tumor cells by NK cells
FPlZl4s 33 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167~1~WARENDS
mediated by an antibody.
The term “complement-dependent cytotoxicity activity”
as used herein refers to ement-dependent cytotoxicity
(CDC)activity”andneanstheactivityofckmagingtargetcells
such as tumor cells by complement mediated by an antibody.
The term “functional fragment of an antibody” as used
herein refers to a partial fragment of an antibody having an
antigen—binding activity, wherein the fragment has a full or
partialfunctrmioftheantibody,includingFab,F(ab’)2,scFv,
and the like. rn1also.includes Fab' iseamonovalent
fragment in a variable region of an antibody obtained by
treatingF(ab’)2underreducingconditions. However,theterm
is not d to these molecules as long as the fragment has
a binding affinity for an antigen. Further, these onal
fragments include not only a fragment obtained by treating a
full—lengthnmleculecflfanantibodyproteinwithen1appropriate
enzyme, but.alsoaaproteiriproducedjjlan.appropriate host cell
using a genetically modified antibody gene.
The term “Fab’” as used herein refers to a monovalent
fragment in a variable region of an antibody obtained by
treating F (ab’ ) 2 under reducing conditions as described above.
r, Fab’ produced using a genetically modified antibody
gene is also included in the Fab’ of the invention.
FP1214s 34 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
The term “epitope” as used herein refers to a partial
e or a partial tertiary structure of B7—H3 to which a
specific anti—B7—H3 antibody binds. The epitope which is a
partial peptide of the above—described B7—H3 can be determined
by a method well known to those skilled in the art such as an
immunoassay, and for example, the following method can be
employed. First,variouspartialstructuresofzm1antigenare
produced. In the production of the l structures , a known
eptide synthesis que can be used. For example,
a series of polypeptides having appropriately reduced lengths
obtained.by sequentially shortening B7—H3 from the C terminus
or N terminus are produced using a c recombination
technique known to those skilled in the art. Thereafter, the
reactivity of an antibody against these polypeptides is
examined and.a recognition site is roughly determined. Then,
peptides having shorter lengths are synthesized and the
reactivity of the antibody with these peptides is examined,
whereby the epitope can be determined. Further, the epitope
which is a l tertiary structure of an n to which
a specific antibody binds can be determined by specifying the
amino acid residues of the n which lie adjacent to the
antibody by X—ray structural analysis.
The phrase “antibodies which bind to the same epitope”
as used herein refers to different antibodies which bind to
a common epitope. If a second antibody binds to a partial
peptide or a partial tertiary structure to which a first
antibody binds, it can be determined that the first antibody
and the second antibody bind to the same epitope. Further,
by confirming that a second antibody competes with a first
antibody for the binding to an antigen (that is, the second
antibody inhibits the binding between the first antibody and
the antigen), it can be determined that the first antibody and
the second antibody bind to the same epitope even if the
icepitopesequenceorstructurehasnotbeendetermined.
Further, when the first antibody and the .antibody bind
to the same epitope and also the first antibody has a special
activity such as an.antitumor activity, it carlbe expected that
the second antibody also has the same activity. Accordingly,
when a second anti—B7—H3 antibody binds to a partial peptide
towhichaafirstanti—B7—H3antibodybinds,itcxnibedetermined
that the first antibody and the second antibody bind to the
same epitope of B7-H3. Further, by confirming that a second
anti—B7—H3 antibody competes with.a first 7—H3 antibody
for the binding to B7—H3, it can be determined that the first
antibody and.the second dy are dies which bind to
the same e of B7—H3.
The term “CDR” as used herein refers to a mentarity
determining region (CDR), and it is known that each heavy and
FP1214s 36 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
light chain of an antibody molecule has three complementarity
determining regions . The CDR is also called the
hypervariable region, and is present in a variable region of
each heavy and light chain of an antibody. It is a site which
has unusually high variability in its primary structure, and
there are three separate CDRs in.the primary structure of each
heavy and light polypeptide chain. In this specification,
with regard to the CDRs of an dy, the CDRs of the heavy
chain are represented by CDRHl, CDRH2, and CDRH3 from the
amino-terminal side of the amino acid sequence of the heavy
chain, and the CDRs of the light chain are represented by CDRLl,
CDRLZ, and CDRL3 from the amino-terminal side of the amino acid
sequence of the light chain. These sites are proximate to one
anotherinthetertiarystructureanddeterminethespecificity
for an antigen to which the antibody binds.
The phrase dization is performed under stringent
conditions" as used herein refers to a process in which
hybridization. is performed under conditions under which
identification can be achievedkn/performing hybridization at
68°C in a cially available hybridization solution
sHyb Hybridization Solution (manufactured by Clontech,
Inc.) or by performing hybridization at 68°C in the presence
of 0.7 to 1.0 M NaCl using a filter having DNA immobilized
thereon, followed by performing washing at 68°C using 0.1 to
s 37 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
2 x SSC solution (1 x SSC solution is composed of 150 mM NaCl
and 15 mM sodium citrate) or under conditions lent
thereto.
1. B7—H3
B7-H3 is a member of the B7 family expressed on
antigen—presenting cells as a costimulatory molecule, and is
considered to act on a receptor on T cells to enhance or suppress
immune activity.
B7—H3 is a protein having a single—pass transmembrane
structure, and the N—terminal extracellular domain of B7-H3
contains two variants. The B7—H3 variant 1 (4Ig—B7—H3)
contains a V—like or C—like Ig domain at two sites, respectively,
aB7—H3 variant2 7—H3)containseaV—like<m:C-like
Ig domain at one site, tively.
As for B7—H3 to be used in the invention, B7—H3 can be
directly purified from B7—H3—expressing cells of a human or
a non—human mammal (such as a rat or a mouse) and used, or a
cell membrane fraction of the above—described cells can be
prepared and used. Further, B7—H3 can be obtained.by in vitro
synthesis thereof or production.thereof inaihost cell through
genetic engineering. In such genetic engineering,
specifically, after B7—H3 cDNA is integrated into a vector
capable of expressing B7—H3 cDNA, B7—H3 can be obtained by
synthesizing it in a solution containing enzymes, ates
FP1214s 38 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
and energy substances required for transcription and
translation, or by expressing B7—H3 in another prokaryotic or
eucaryotic ormed host cell
The nucleotide sequence of an open reading frame (ORF)
of a human B7—H3 variant 1 gene is ented by SEQ ID NO:
in the Sequence Listing, and the amino acid sequence f
isrepresentedknzSEQIHDNO:6zu1theSequenceIdsting. r,
the sequences of SEQ ID NOS: 5 and 6 are shown in Fig. 13.
ThenucleotidesequenceofenioRFofeahumanB7—H3variant
2 gene is represented by SEQ ID NO: 9 in the Sequence Listing,
and the amino acid sequence thereof is represented by SEQ ID
NO: 10 in the Sequence Listing. r, the sequences of SEQ
ID NOS: 9 and 10 are shown in Fig. 14.
Further, a protein which consists of an amino acid
sequence wherein one or several amino acids are substituted,
deleted and/or added.in any of the above—described amino acid
sequences of B7—H3 and also has a biological activity
equivalent to that of the protein is also included in B7-H3.
Mature human B7-H3 variant 1 from which the signal
sequencehasbeenremovedcorrespondstoaniaminoacidsequence
consisting of amino acid residues 27 to 534 of the amino acid
sequence represented by SEQ ID NO: 6. Further, mature human
B7—H3 variant 2 from which the signal sequence has been d
corresponds toauiamino acid sequence consisting of amino acid
FP1214S 39 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
residues 27 to 316 of the amino acid sequence represented by
SEQ ID NO: 10.
In the B7—H3 variant 1, the respective domains are
t in the order of Ing, IgCl, IgV2, and IgCZ from the
N terminus, and in SEQ ID NO: 6 in the Sequence Listing, Ing
corresponds to amino acid numbers 27 to 139, IgCl corresponds
to amino acid numbers 140 to 244, IgV2 corresponds to amino
acid numbers 245 to 357, and IgC2 corresponds to amino acid
numbers 358 to 456. Further, in the B7—H3 variant 2, the
respective domains are present in the order of Ing and IgC2
from the N terminus and in SEQ ID NO: 10 in the Sequence Listing,
Ing corresponds to amino acid numbers 27 to 140 and IgC2
corresponds to amino acid numbers 141 to 243.
B7—H3 CDNA can be obtained.by, for example, a led
PCR method in which a polymerase chain reaction (hereinafter
referred to as “PCR”) is performed using a cDNA library
expressing B7—H3 cDNA. as a template and. primers which
specificallyamplifyB7—H3cDNA(Saiki,RAIL, etal.,Science,
(1988) 239, 487—49). Incidentally, a polynucleotide which
izes to a cleotide consisting of a nucleotide
ce complementary‘to'the1m1cleotide sequence represented
by SEQ ID NO: 5 or 9 in the Sequence Listing under stringent
ions and encodes a protein.having a biological activity
equivalent to that of B7—H3 is also included in B7—H3 cDNA.
Further,apwlynucleotidewhichij;asplicingvarianttranscribed
from the human or mouse B7—H3 locus or a cleotide which
hybridizes to ea polynucleotide consisting' of a. nucleotide
sequence complementary thereto under stringent conditions and
s a protein having a ical activity equivalent to that
of B7—H3 is also included in B7—H3 CDNA.
Further, a protein which consists of an amino acid sequence
n one or several amino acids are substituted, deleted or
added in the amino acid sequence represented by SEQ ID NO: 6 or
in the Sequence Listing or an amino acid ce obtained
by removing the signal sequence from either of these sequences
and.has a biological activity equivalent to that of B7—H3 is also
included in B7—H3 . Further, a protein which consists of an amino
acid sequence encoded.by a splicing t transcribed.from the
human or mouse B7—H3 locus or an amino acid sequence wherein one
or several amino acids are substituted, d or added in the
aboveaminoacidsequenceandhasaabiologicalactivityequivalent
to that of B7—H3 is also included in B7—H3.
2. Production of anti—B7—H3 antibody
The antibody against B7-H3 of the invention can be
obtained by immunizing an animal with B7—H3 or an arbitrary
polypeptide selected from the amino acid sequence of B7—H3,
and collecting and purifying the antibody produced in vivo
according to common procedures. The biological species of
B7—H3 to be used as an antigen is not limited to being human,
and an animal can be immunized with B7—H3 derived from an animal
other than humans such as a mouse or a rat. In this case, by
examining the cross—reactivity’between.an antibody binding to
the ed heterologous B7~H3 and human B7—H3, an antibody
applicable to a human e can be selected.
Further, a monoclonal antibody can be obtained from a
hybridoma established by fusing antibody—producing cells
which produce an dy against B7—H3 with myeloma cells
according to known methods (for example, Kohler and Milstein,
Nature, (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal
dies, pp. 365—367, Plenum Press, N.Y. (1980)).
Incidentally, B7—H3 to be used as an antigen can be
obtained by expressing the B7—H3 gene in a host cell using
genetic enginering.
Specifically; a vector capable of expressing the B7—H3
gene:is produced, and the resulting vector is transfected.into
a host cell to express the gene, and then, the expressed B7—H3
is purified. after, a method of ing an antibody
against B7—H3 will be specifically described.
FP1214S 42 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
(1) Preparation of antigen
Examples of the antigen to be used for ing the
anti—B7—H3 antibody include B7—H3, a polypeptide consisting
of a partial amino acid sequence comprising at least 6
consecutive amino acids of B7—H3, and a derivative obtained
by adding a given amino acid sequence or r thereto.
B7—H3 can be purified directly from human tumor tissues
or tumor cells and used. Further, B7-H3 can be obtained by
synthesizing it in vitro or by producing it in a host cell by
genetic engineering.
With respect to such genetic engineering, specifically,
after B7—H3 cDNA is integrated into a vector capable of
expressing B7—H3 cDNA, B7—H3 can be obtained by synthesizing
it in a solution containing enzymes, substrates and energy
substances required for transcription and translation, or by
expressing B7—H3 in another yotic or eucaryotic
transformed host cell.
Further, the n can also be obtained as a secretory
protein by expressing a fusion protein obtained by ligating
theextracellulardomahiofB7—H3,whichisaamembraneprotein,
to the constant region of an antibody in an appropriate
host-vector system.
FP1214s 43 810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
B7-H3 cDNA can be obtained by, for e, a so—called
PCR method in which a rase chain reaction (hereinafter
referred to as “PCR”) is performed using a cDNA library
expressing B7—H3 cDNA. as a template and primers which
specifically amplify B7—H3 cDNA (see Saiki, R. K., et al.,
Science, (1988) 239, pp. 487—489).
As the systen1for in vitro synthesis of the polypeptide,
for example, Rapid Translation System (RTS) ctured by
Roche stics, Inc. can be exemplified, but it is not
limited thereto.
Examples of prokaryotic host cells include Escherichia
coli and Bacillus is. In order to transform the host
cells with a target gene, the host cells are transformed by
a plasmid vector comprising a on, i.e., a replication
origin derived from a species compatible with the host, and
a regulatory sequence. Further, the vector preferably has a
sequence capable of imposing phenotypic selectivity on the
transformed cell.
Examples of eucaryotic host cells include vertebrate
cells,insectcells,andyeastcells. .Asthevertebratecells,
for example, simian COS cells (Gluzman, Y., Cell, (1981) 23,
FP1214S 44 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
pp. 175—182, ATCC CRL-l650), murine fibroblasts NIH3T3 (ATCC
No. CRL—l658), and dihydrofolate reductase-deficient strains
(Urlaub,(3.andChasin,Ih A.,Proc.Natl.Acad.Sci.USA(1980)
77, pp. 4126—4220) oftihinese hamster ovarian cells (CHO<:ells;
ATCC: CCL—6l); and the like are often used, however, the cells
are not limited thereto.
Thethusobtainedtransformant<xu1beculturedaccording
to common procedures, and by culturing the transformant, a
target polypeptide is ed intracellularly or
extracellularly.
A suitable medium to be used for culturing can be selected
from s commonly used culture media depending on the
employed host cells. If Escherichia coli is ed, for
example, an LB medium supplemented.with an antibiotic such as
ampicillin or IPMG as needed can be used.
A recombinant protein produced intracellularly or
extracellularlykmzthetransformantthroughsuchculturingcan
be separated and ed by any of various known separation
methods utilizing the physical or chemical properties of the
Specific examples of the methods include treatment with
FP1214S 45 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
a common protein precipitant, ultrafiltration, various types
of liquid chromatography such as molecular sieve
chromatography (gel filtration), adsorption chromatography,
ion exchange chromatography, and affinity chromatography,
dialysis, and a combination thereof.
Further, by attaching a tag of six histidine residues
to a recombinant protein to be expressed, the n can be
efficiently purified with a nickel affinity column.
Alternatively, by attaching the IgG Fc region toaarecombinant
protein to be expressed, the protein can be efficiently
purified with a protein A column.
By combining the above—described methods, a large amount
of a target polypeptide can be easily produced in high yield
and high purity.
(2) tion of anti—B7-H3 monoclonal antibody
Examples of the antibody specifically binding to B7—H3
include a monoclonal antibody specifically binding to B7—H3,
and alnethod of ing such an antibody is described below.
The production of a Inonoclonal antibody generally
requires the ing operational steps of:
(a) ing a biopolymer to be used as an n;
FP1214S 46 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
(b) ing antibody—producing cells by immunizing an
animal by injection of the antigen, collecting the blood,
assaying its antibody titer to determine when the spleen is
excised;
(c) preparing myeloma cells nafter referred to as
"myeloma");
(d) fusing the antibody—producing cells with the
myeloma;
(e) ing a group of hybridomas producing a desired
antibody;
(f) dividing the hybridomas into single cell clones
(cloning);
(g) optionally, culturing the hybridoma or rearing an
animal implanted with the hybridoma for producing a large
amount of a monoclonal antibody;
(h) examining the thus producedlnonoclonal antibody for
biological activity and binding specificity, or examining the
properties of the dy as a labeled antibody reagent; and
the like.
Hereinafter, the method. of producing a Inonoclonal
antibodyvnjj_bedescribedjjldetailfollowingtflmaabovesteps,
however, the method is not limited thereto, and, for example,
antibody-producing cells other than spleen cells and a
can be used.
F91214s 47 BlOOZB/Eng trans of PCT spec/26.9.13
4426167WARENDS
(a) Purification of antigen
Astheantigen,B7-H3preparafibythenethodasCkscribed
above or a partial peptide thereof can be used.
Further, a membrane fraction prepared from recombinant
cells sing B7—H3 or the recombinant cells expressing
B7—H3 themselves, and also a partial peptide of the protein
of the invention chemically synthesized by a method known to
those skilled in the art can also be used as the antigen.
(b) Preparation of antibody—producing cells
The antigen obtained in step (a) is mixed with an adj uvant
such as Freund’s complete or incomplete adjuvant or um
potassium e and the resulting mixture is used as an
immunogen to immunize an experimental animal. As the
experimental animal, any animal used in a known oma
production method can be used without any trouble.
Specifically,forexample,anwuse,aixng agoat,sheep,cattle,
a horse, or the like can be used. However, fronlthe int
of ease of availability of myeloma cells to be fused with the
ted antibodynproducing cells, a mouse or 23 rat is
preferably used as the animal to be immunized.
Further, the strain of mouse or rat to be used is not
FP1214s 48 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
particularly d, and in the case of alnouse, for example,
various strains such asZL AKR, BALB/c, BDP, BA, CE, C3H, 57BL,
C57BL, C57L, DBA, FL, HTH, HTl, LP, NZB, NZW, RF, R III, SJL,
SWR, WB, and 129 and the like can be used, and in the case of
a rat, for example, Wistar, Low, Lewis, Sprague Dawley, AC1,
BN, Fischer and the like can be used.
These mice and rats are commercially ble from
breeders/distributors of experimental animals, for example,
CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.
Amongthese,jjlconsideration<1fcompatibility<flffusing
with myeloma cells described below, in the case of a mouse,
BALB/c strain, and in the case of a rat, Wistar and Low strains
are particularly preferred as the animal to be immunized.
Further, in eration.of antigenic homology’between
humans and mice, it is also preferred to use a mouse having
decreased biological function to remove autoantibodies, that
is, a mouse with an autoimmune disease.
The age of such mouse or rat at the time of immunization
is preferably 5 to 12 weeks of age, more ably 6 to 8 weeks
of age.
InordertojflmmnizeananimalwithB7—H3<n:arecombinant
FP1214s 49 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
thereof, for example, a known method described in detail in,
for example, Weir, D. M., Handbook of Experimental Immunology
Vol. I. II. III., Blackwell ific Publications, Oxford
(1987), Kabat, E. A. and Mayer, M. M., mental
Immunochemistry, Charles (3 Thomas Publisher Springfield,
is (1964) or the like can be used.
Among these immunization methods, a preferred specific
method of the invention is, for example, as follows.
That is, first, a membrane protein fraction serving as
the antigen or cells caused to express the antigen is/are
intradermallgzor intraperitoneally'administeredtx:an.animal.
However, the combination of both routes of
administration is preferred for increasing the immunization
efficiency, and when intradermal administration is performed
in the first half and intraperitoneal administration is
med in the latter half or only at the last dosing, the
immunization efficiency can be particularly increased.
The administration schedule of the antigen. varies
depending on the type of animal to be immunized, individual
differences or the like. However, in general, an
administration schedule in which the number of times antigen
is stered is 3 to 6 times and the dosing interval is 2
FP1214s 50 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
tr>6weeksiispreferred,andaniadministrationscheduleghiwhich
the number of times antigen is administered is 3 to 4 times
and the dosing interval is 2 to 4 weeks is more preferred.
Further, the dose of the antigen varies depending on the
type of , individual differences or the like, however,
the dose is generally set to 0.05 to 5 mg, preferably about
0.1 to 0.5 mg.
A booster immunization is performed 1 11) 6 weeks,
preferably 2 to 4 weeks, more preferably 2 to 3 weeks after
the administration of the antigen as described above.
The dose of the antigen at the time of performing the
booster immunization varies depending on the type or size of
animal or the like, however, in the case of, for e, a
mouse, the dose is lly set to 0.05 to 5 mg, preferably
0.1 to 0.5 mg, more preferably about 0.1 to 0.2 mg.
Spleen cells or lymphocytes including
antibody—producing cells are aseptically removed from the
immunized animal 1 to 10 days, preferably 2 to 5 days, more
preferably 2 to 3 days after the booster immunization. At this
time, the antibody titer is measured, and if an animal having
a sufficiently increased antibody titer is used as a supply
FP1214s 51 810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
source of the antibody—producing cells, the subsequent
procedure can be carried out more efficiently.
Examples of the method of measuring the dy titer
to be used.here include an RIAInethod and an ELISAJnethod, but
the method is not limited thereto.
For e, if an ELISA method is ed, the
ement of the antibody titer in the invention can be
carried out according to the procedures as described below.
First, a purified or partially purified antigen is
adsorbed to the surface of a solid phase such as a 96—well plate
for ELISA, and the surface of the soliriphase havingIKDantigen
adsorbed thereto is covered with a protein unrelated to the
antigen such as bovine serunlalbumin (hereinafter referred to
as "BSA"). After washing the surface, the surface is brought
intocontactwithxaserially—dilutedsample(forexample,nmuse
serum)aseaprimaryantibodyixaallowifluaantibodyijlthesample
to bind to the antigen.
r, as a secondary antibody, an antibody labeled
with an enzyme against a mouse antibody is added and is allowed
to bind to the mouse antibody. After washing, a substrate for
the enzyme is added and a change in absorbance which occurs
FP1214s 52 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
due to color development induced. by degradation. of the
substrate or the like is measured and the antibody titer is
calculated based on the measurement.
The separation of the antibody—producing cells he
spleen cells or lymphocytes of the immunized animal can be
carried out according to a known method (for example, Kohler
et al., Nature (1975), 256, p. 495; Kohler et al., Eur. J.
Immunol. (1977), 6, p. 511; Milstein et al., Nature (1977),
266, p. 550; Walsh, Nature (1977), 266, p. 495). For e,
in the case of spleen cells, a general method in which the
antibody-producing cells are separated by homogenizing the
spleen to obtain the cells through filtration.withaastainless
steel mesh and suspending the cells in Eagle's Minimum
Essential Medium (MEM) can be employed.
(c) Preparation of myeloma cells (hereinafter referred to as
"myeloma")
The myeloma cells to be used for cell fusion are not
particularly limited and suitable cells can be ed from
known cell lines. However, in consideration of convenience
when a hybridoma is selected_from.fused cells, it is red
to use an HGPRT (hypoxanthine—guanine oribosyl
transferase) deficient strain whose selection procedure has
been ished.
FP1214s 53 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
More ically, examples of such an HGPRT-deficient
strain include X63—Ag8(X63), NSl—ANS/l(NSl),
P3X63-Ag8.Ul(P3Ul), X63—Ag8.653(X63.653), SP2/O—Agl4(SP2/O),
MPCll-45.6TG1.7(45.6TG), FO, Sl49/5XXO, and BU.l.derived.from
mice; 210.RSY3.Ag.l.2.3(Y3) derived from rats; and
U266AR(SKO-OO7), GMlSOOGTG—A12(GM1500), UC729-6,
LICR—LOW—HMy2(HMy2) and 8226AR/NIP4—1(NP41) derived from
humans. TheseHGPRT-deficientstrainsareayailablefrom,for
example, the American Type Culture Collection (ATCC) or the
like.
[0079}
These cell strains are subcultured in an appropriate
medium such as an uanine medium [a medium obtained by
adding 8—azaguanine to an RPMI 1640 medium mented with
glutamine, 2—mercaptoethanol, gentamicin, and fetal bovine
serum (hereinafter referred to as "FBS")], Iscove’s Modified
Dulbecco’s Medium (hereinafter referred to as "IMDM"), or
Dulbecco’s Modified Eagle Medium (hereinafter referred to as
"DMEM"). In this case, 3 to 4 days before performing cell
, the cells are tured in a normal medium [for
example,anASFlO4medium(manufacturedbijinomotoCo.,Lth
containing 10% PBS] to ensure not less than 2 x 107 cells on
the day of cell fusion.
(d) Cell fusion
Fusion between the antibody-producing cells and the
myeloma cells can be appropriately performed according to a
known method (Weir, D. M. ok of Experimental Immunology
Vol. I. II. III., Blackwell Scientific Publications, Oxford
(1987), Kabat, E. A. and Mayer, M. M., Experimental
chemistry, Charles C Thomas Publisher, Springfield,
Illinois(1964),etc.),underconditionssuchthatthesurvival
rate of cells is not ively reduced.
As such a method, for example, a chemical method in which
the antibody—producing cells and the a cells are mixed
in a solution containing a polymer such as polyethylene glycol
at a high concentration, a physical method using electric
stimulation, or the like can be used. Among these methods,
aspecificexampleofthechemicalmethodisasckwcribedbelow.
That is, in the case where polyethylene glycol is used
in.the solution containingeapolymer ateahigh concentration,
the antibody—producing cells and the myeloma cells are mixed
in a solution of polyethylene glycol having a molecular weight
of 1500 to 6000, more preferably 2000 to 4000 at a temperature
offrmn30t040°C,preferablyfrom3511>38°CforiltolOnunutes,
ably 5 to 8 minutes.
(e) Selection of a group of hybridomas
FP1214s 55 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
The method of selecting hybridomas obtained by the
described cell fusion is not particularly limited.
Usually, an HAT (hypoxanthine, aminopterin, thymidine)
selection method (Kohler et al., Nature (1975), 256, p. 495;
Milstein et al., Nature (1977), 266, p. 550) is used.
This method is effective when hybridomas are obtained
using the myeloma cells of an deficient strain which
cannot survive in the presence of aminopterin.
That is, by culturing unfused cells and hybridomas in
an HAT medium, only hybridomas resistant to terin are
selectively allowed to survive and proliferate.
(f) Division into single cell clone (cloning)
As a cloning method for hybridomas, a known.method such
as a methylcellulose , a soft agarose method, or a
limitingdilutionnethod(xn1beused(see,forexample,Barbara,
B. M. and Stanley, M. 8.: Selected Methods in Cellular
Immunology, W. H. Freeman and.Company, San Francisco (1980)).
Among these s, particularly, a three-dimensional
culture method such as a methylcellulose method is preferred.
For example, the group of hybridomas produced by cell fusion
is suspended in alnethylcellulose mediunlsuch as ClonaCell—HY
Selection Medium D actured by StemCell Technologies,
FP1214s 56 WGA/PN810023/Eng trans of PCT 6.9.13
4426167-1—WARENDS
inc., #03804) and cultured. Then, the formed hybridoma
colonies are collected, whereby monoclonal hybridomas can be
obtained. The collected respective hybridoma colonies are
cultured, and a hybridoma which has been confirmed to have a
stable dy titer in an ed hybridoma culture
supernatant is selected as a B7—H3 monoclonal
antibody—producing hybridoma strain.
Examples of the thus established hybridoma strain
e B7—H3 hybridoma M30. Incidentally, in this
specification, an antibody produced by the B7—H3 oma M30
is referred to as “M30 antibody” or simply “M30”.
The heavy chain of the M30 antibody has an amino acid
sequence representemiby SEQ ID NO: 51 in the Sequence Listing.
Further, the light chain of the M30 antibody has an amino acid
sequence ented by SEQ ID NO: 53 in the Sequence Listing.
Incidentally, in the heavy chain amino acid sequence
representeciby SEQ IDDKX 51 in the ce Listing, the amino
acid sequence consisting of amino acid residues 1 to 19 is a
signal sequence, the amino acid sequence consisting of amino
acid es 20 to 141 is a variable region, and the amino
acid sequence consisting of amino acid residues 142 to 471 is
a constant region. r, in the light chain amino acid
sequence represented by SEQ ID NO: 53 in the Sequence Listing,
the amino acid sequence consisting of amino acid residues 1
FP1214S 5'7 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
tc>22 is a signal sequence, the amino acid sequence consisting
of amino acid residues 23 to 130 is a le region, and the
amino acid sequence consisting of amino acid residues 131 to
235 is a constant region.
The heavy chain amino acid sequence represented by SEQ
ID NO: 51 in the Sequence Listing is encoded by a nucleotide
sequence representeciby SEQ ID NO: 50 in the Sequence Listing.
In the nucleotide sequence represented by SEQ ID NO: 50 in the
Sequence Listing, the nucleotide ce consisting of
nucleotides 1 to 57 encodes the heavy chain signal sequence
of the antibody, the nucleotide sequence consisting of
nucleotides 58 to 423 encodes the heavy chain variable region
of the antibody, and the nucleotide sequence ting of
tides 424 to 1413 encodes the heavyrchain.constant.region
of the antibody.
The light chain amino acid sequence represented by SEQ
ID NO: 53 in the Sequence Listing is encoded by a tide
ce represented.by SEQ ID NO: 52 in the Sequence Listing.
In the nucleotide sequence represented by SEQ ID NO: 52 in the
Sequence Listing, the nucleotide sequence consisting of
nucleotides 1 to 66 encodes the light chain signal sequence
of the antibody, the nucleotide sequence ting of
nucleotides 67 to 390 encodes the light chain variable region
FP1214s 58 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
of the dy, and the nucleotide sequence consisting of
nucleotides 391 to 705 encodes the light chain constant region
of the antibody.
(g) Preparation.ofxnonoclonal antibody by culturing oma
By culturing the thus selected hybridoma, a monoclonal
antibody can be efficiently obtained. However, prior to
culturing, it.is preferredTx>perform.screening ofaahybridoma
which produces a target monoclonal antibody.
In such screening, a known method can be employed.
The measurement of the antibody titer in the invention
can.be carried out by, for example, an ELISA.method.explained
in item (b) described above.
Thehybridomaobtainedku/thenethoddescribedabovecan
be stored in a frozen state in liquid nitrogen or in a freezer
at —80°C or below.
After completion of cloning, the mediunlis changed from
an.HT1nediun1tc>a lnedium, and the hybridoma is cultured.
scale culture is performed by rotation culture
using a large culture bottle or by spinner culture. From the
S 59 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
supernatant ed by the large—scale culture, almonoclonal
antibody which specifically binds to the protein of the
invention can be obtained by purification using methods known
to those skilled in the art such as gel filtration.
r, the hybridoma is injected into the nal
cavity of a mouse of the same strain as the oma (for
example, the above—described BALB/c) or a Nu/Nu mouse to
proliferate the hybridoma, whereby the ascites containing a
large amount of the monoclonal antibody of the invention can
be obtained.
In cases where the hybridoma is administered to the
abdominal cavity, if a mineral oil such as
2,6,10,14—tetramethyl pentadecane (pristane) is administered
3 to 7 days prior thereto, a larger amount of the ascites can
be obtained.
mple,animmunosuppressantispreviouslyinjected
into the nal cavity of a mouse of the same strain as the
hybridoma to inactivate T cells. 20 days thereafter, 106 to
lO7hybridoma clone cells are suspended.inaaserum—free.medium
(0.5 ml), and the suspension is administered to the abdominal
cavity of the mouse. In general, when the abdomen is expanded
and.filled.with.the ascites, the ascites is collected from the
FP1214s 6O WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
mouse. Bythisnethod,thenwnoclonalantibodycanbecbtained
at a concentration which is about 100 times or more higher than
that in the culture solution.
Thelnonoclonal antibody obtaineciby the above—described
method can be purified by a1nethod described in, for example,
Weir, D. M.: Handbook of Experimental Immunology Vol. I, II,
III, ell Scientific Publications, Oxford (1978).
The thus obtained.monoclonal antibody has high n
specificity for B7-H3.
(h) Assay of monoclonal antibody
The isotype and ss of the thus obtainedlnonoclonal
antibody can be determined as follows.
First, examples of the identification.method.include an
Ouchterlony method, an ELISA method, and an RIA method.
An Ouchterlony method is , but when the
concentratbmiofthenmnoclonalantibodyisiknn acondensation
operation is required.
On the other hand, when an ELISA.method or an RIAInethod
is used, by directly reacting the culture supernatant with an
s 61 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
antigen—adsorbed solid phase and using antibodies
corresponding to various types of immunoglobulin isotypes and
subclasses as secondary antibodies, the isotype and subclass
of the monoclonal dy can be identified.
In addition, as a simpler method, a commercially
available identification kit (for example, Mouse Typer Kit
manufactured by Bio—Rad Laboratories, Inc.) or the like can
also be used.
Further,thequantitativedeterminationofaaproteincan
be performed by the Folin Lowry method and a method of
calculation based on the absorbance at 280 nm [1.4 (OD 280)
= Immunoglobulin 1 mg/ml].
Further,evenwhenthenmnoclonalantibodyisseparately
and independently obtained by performing again the steps of
(a) to (h) in (2), it is le to obtain an antibody having
a cytotoxic activity equivalent to that of the M30 antibody.
As one example of such an dy, an antibody which binds
to the same epitope as the M30 antibody can be exemplified.
The:M30 recognizes an.epitope in the IgCl or IgC2 , which
is a domain in the B7—H3 extracellular domain, and binds to
the IgCl domain or the IgC2 domain or both. Therefore, as the
epitopeforiflmaM30antibody,particularly,eniepitopepmesent
FP1214S 62 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
in the IgCl or IgCZ domain of B7—H3 can be exemplified. If
anewlyproducednmnoclonalantibodybindstoaapartialpeptide
or a partial ry structure to which the M30 antibody binds,
it can.be determined that the:monoclonal antibody binds to the
same epitope as the M30 antibody. Further, by confirming that
thelnonoclonal antibody competes with thelM3O antibody for the
binding to B7—H3 (that is, the monoclonal antibody inhibits
the binding between the M30 antibody and B7—H3), it can be
determined that the monoclonal antibody binds to the same
e as the M30 antibody even if the ic epitope
sequence or ure has not been determined. When it is
confirmed that the monoclonal antibody binds to the same
e as the M30 antibody, the nwnoclonal antibody is
strongly ed to have a cytotoxic activity equivalent to
that of the M30 antibody.
(3) Other antibodies
The dy of the invention includes not only the
above—described.monoclonal antibody against B7—H3 but also a
recombinant antibody obtained by artificial modification for
the purpose of decreasing heterologous antigenicity to humans
such as a chimeric antibody, a humanized antibody and a human
antibody. These antibodies can. be produced using known
methods.
FPlZl4s 63 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
As such a chimeric antibody, an antibody in which
antibody le and constant regions are derived from
different species, for example, a chimeric antibody in which
a mouse— or rat-derived dy variable region.is ted
tx>ahuman—derivedconstantregioncxulbeexemplified(seeProc.
Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). A chimeric
antibody derived from a mouse anti—human B7—H3 antibody M30
is an antibody consisting of a heavy chain comprising a heavy
chainvariableregioncnfwhidntheaminoacidsequenceconsists
of amino acid residues 20 to 141 of SEQ ID NO: 51 and a light
chain comprising a light chain le region of which the
amino acid sequence consists of amino acid residues 23 to 130
of SEQ ID NO: 53, and may have an arbitrary human-derived
constant region. As one example of such a chimeric antibody,
an.antibody consisting ofeaheavy'chairlof which the amino acid
sequence consists of amino acid residues 1 to 471 of SEQ ID
NO: 63 in the Sequence Listing and a light chain of which the
amino acid sequence ts of amino acid residues 1 to 233
of SEQ ID NO: 59 in the Sequence Listing can be exemplified.
Incidentally, in the heavy chain sequence represented.by SEQ
ID NO: 63 in the Sequence Listing, the amino acid sequence
ting of amino acid residues 1 to 19 is a signal sequence,
the amino acid sequence ting of amino acid residues 20
to 141 is a variable region, and the amino acid sequence
consisting of amino acid residues 142 to 471 is a constant
FP1214s 64 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
region. Further, in the light chain sequence represented by
SEQIHDNO:59:UItheSequenceListing,the aminoacidsequence
consisting of amino acid residues].to 20i53a signal sequence,
the amino acid sequence consisting of amino acid residues 21
to 128 is a variable region, and the amino acid sequence
consisting of amino acid residues 129 to 233 is a constant
region.
The heavy chain amino acid sequence represented by SEQ
ID NO: 63 in the Sequence Listing is encoded by a nucleotide
ce represented by SEQ ID NO: 62 in the ce Listing.
In the nucleotide sequence represented by SEQ ID NO: 62 in the
Sequence Listing, the nucleotide sequence ting of
nucleotides 1 to 57 encodes the heavy chain signal sequence
of the antibody, the nucleotide sequence ting of
nucleotides 58 to 423 encodes the heavy chain variable region
of the antibody, and the nucleotide sequence consisting of
nucleotides 424 to 1413 encodes thelneav570haijlconstant region
of the dy.
The light chain amino acid sequence represented by SEQ
ID NO: 59 in the Sequence Listing is encoded by a nucleotide
sequence represented.by SEQ ID NO: 58 in the Sequence Listing.
In the nucleotide ce ented by SEQ ID NO: 58 in the
Sequence Listing, the nucleotide sequence consisting of
FP1214s 65 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
nucleotides 1 to 60 encodes the light chain signal sequence
of the antibody, the nucleotide sequence consisting of
nucleotides 61 to 384 encodes the light chain variable region
of the antibody, and the nucleotide sequence consisting of
nucleotides 385 to 699 encodes the light chain.constant region
of the antibody.
As such a humanized dy, an antibody obtained by
ating only a complementarity determining region (CDR)
into a human—derived antibody (see Nature (1986) 321, pp.
522-525), and an antibody obtained by grafting a part of the
amino acid residues of the framework as well as the CDR sequence
to a human antibody by'a CDR—grafting method (WO 90/07861) can
be exemplified.
However, the humanized antibody d from the M30
antibody is not limited to a ic humanized antibody as
long as the humanized antibody has all 6 types of CDR sequences
of the M30 antibody and has an antitumor activity.
ntally, the heavy chain variable region of the M30
dy has CDRHl (NYVMH) consisting of the amino acid
sequence represented by SEQ ID NO: 92 in the Sequence Listing,
CDRHZ (YINPYNDDVKYNEKFKG) consisting of the amino acid
sequence representeclby SEQ ID NO: 93 in the Sequence Listing,
and CDRH3 (WGYYGSPLYYFDY) consisting of the amino acid
s 66 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
sequence represented by SEQ ID NO: 94 in the Sequence Listing.
Further, the light chain variable region of the M30 dy
has CDRLl (RASSRLIYMH) consisting of the amino acid sequence
represented by SEQ ID NO: 95 in the Sequence Listing, CDRL2
(ATSNLAS) ting of the amino acid sequence represented
by SEQ IDIMD: 96jJ1the Sequence Listing, and CDRL3 (QQWNSNPPT)
consisting of the amino acid sequence ented by SEQ ID
NO: 97 in the Sequence Listing.
As an example of the humanized antibody of mouse antibody
M30, an arbitrary ation of a heavy chain comprising a
heavy chain variable region consisting of any one of (1) an
amino acid sequence consisting of amino acid residues 20 to
141 of SEQ ID NO: 85, 87, 89, or 91 in the Sequence Listing,
(2) an amino acid sequence having a homology of at least 95%
or more with the amino acid ce (1) described above, and
(3) an amino acid sequence wherein one or several amino acids
in the amino acid sequence (1) described above are deleted,
substituted or added and a light chain comprising a light chain
variable region consisting of any one of (4) an amino acid
sequence consisting of amino acid residues 21 to 128 of SEQ
ID NO: 71, 73, 75, 77, 79, 81, or 83 in the Sequence Listing,
(5) an amino acid sequence having a gy of at least 95%
or more with the amino acid sequence (4) described above, and
(6) an amino acid ce wherein one or several amino acids
FP1214s 6'7 WGA/PNBIOOZB/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
in the amino acid sequence (4) described above are deleted,
substituted or added can be exemplified.
ntally, the term “several” as used herein refers
toltolO, lto9, lt08, 11:07, lt06, 11:05, 11:04:,
1 to 3, or 1 or 2.
As the amino acid substitution in this specification,
a conservative amino acid substitution is preferred. The
conservative amino acid.substitution.refers to a substitution
occurring within a group of amino acids related to amino acid
side . red amino acid groups are as follows: an
acidic group (aspartic acid and glutamic acid); a basic group
(lysine,arginine,andhistidine);aIKHkpolargroup(alanine,
valine, leucine, isoleucine, proline, phenylalanine,
methionine, and tryptophan); and an uncharged polar family
(glycine,asparagine,glutamine,cysteine,serine,threonine,
and tyrosine). More preferred amino acid groups are as
follows: an tic hydroxy group (serine and threonine);
an amide—containing group (asparagine and ine); an
aliphatic group (alanine, valine, e, and isoleucine);
and an aromatic group (phenylalanine, tryptophan, and
tyrosine). Such an amino acid substitution is preferably
performed.within a group which does not impair the properties
of a substance having the original amino acid sequence.
As an antibody which has a preferred combination of a
heavy chain and a light chain described above, an antibody
consisting of a heavy chain comprising a heavy chain variable
region consisting of an amino acid sequence ting of amino
acidresidues20tx>1410fSEQIDIKh 85andalightchaincomprising
alightchainvariableregionconsistingofenlaminoacidsequence
consisting of amino acid residues 21 to 128 of SEQ ID NO: 71;
an antibody ting of a heavy chain.comprising a heavy chain
variable region consisting of an amino acid sequence consisting
of amino acid residues 20 to 141 of SEQ ID NO: 85 and a light
chain comprising a light chain variable region consisting of an
amino acid sequence consisting of amino acid residues 21 to 128
of SEQ ID NO: 73; an antibody consisting of a heavy chain
sing a heavy chain variable region consisting of an amino
acid sequence consisting of amino acid residues 20 to 141 of SEQ
ID NO: 85 and a light chain comprising a light chain variable
region consisting of an amino acid ce consisting of amino
acid residues 21 to 128 of SEQ ID NO: 75; an antibody consisting
of a heavy chain comprising a heavy chain variable region
consisting of an amino acid sequence consisting of amino acid
residues 20 to 141 of SEQ ID NO: 85 and a light chain comprising
a light chain variable region consisting of an amino acid
sequence consisting of amino acid residues 21 to 128 of SEQ
ID NO: 77; an antibody consisting of a heavy chain comprising
a heavy chain variable region ting of an amino acid
sequence consisting of amino acid residues 20 to 141 of SEQ
ID NO: 85 and a light chain comprising a light chain variable
region consisting of an amino acid ce consisting of amino
acid es 21 to 128 of SEQ ID NO: 79; an antibody consisting
of a heavy chain comprising a heavy chain variable region
consisting of an amino acid sequence consisting of amino acid
residues 20 to 141 of SEQ ID NO: 85 and a light chain comprising
a light chain variable region consisting of an amino acid
sequence consisting of amino acid residues 21 to 128 of SEQ
ID NO: 81; an antibody consisting of a heavy chain comprising
a heavy chain variable region ting of an amino acid
sequence including amino acid residues 20 to 141 of SEQ ID NO:
85 and a light chain comprising a light chain variable region
consisting of an amino acid sequence consisting of amino acid
residues 21 to 128 of SEQ ID NO: 83; an antibody consisting
of a heavy chain comprising a heavy chain variable region
consisting of an amino acid sequence consisting of amino acid
residues 20 to 141 of SEQ ID NO: 91 and a light chain comprising
a light chain variable region consisting of an amino acid
sequence consisting of amino acid residues 21 to 128 of SEQ
ID NO: 71; an antibody consisting of a heavy chain comprising
a heavy chain le region consisting of an amino acid
sequence consisting of amino acid es 20 to 141 of SEQ
ID NO: 91 and a light chain comprising a light chain variable
regionconsistingofeniaminoacidsequenceconsistingofamino
acid residues 21 to 128 of SEQ ID NO: 73; an antibody consisting
FP1214s 7O WGA/PN810023/Eng trans of PCT 6.9.13
4426167—1-WARENDS
of a heavy chain comprising a heavy chain variable region
consisting of an amino acid sequence consisting of amino acid
es 20 to 141 of SEQ ID NO: 91 and a light chain comprising
a light chain variable region consisting of an amino acid
sequence consisting of amino acid residues 21 to 128 of SEQ
ID NO: 75; and an antibody ting of 21 heavy chain
comprising a heavy chain variable region consisting of an amino
acid sequence ting of amino acid residues 20 to 141 of
SEQ ID NO: 91 and a light chain comprising a light chain variable
region consisting of an amino acid sequence consisting of amino
acid residues 21 to 128 of SEQ ID NO: 77 can be exemplified.
As an antibody which has a more preferred combination of a heavy
chain and a light chain described above,
an antibody consisting of a heavy chain consisting of an amino
acid sequence ented by amino acid numbers 20 to 471 in
SEQ ID NO: 85 and a light chain consisting of an amino acid
sequence represented by amino acid numbers 21 to 233 in SEQ
ID NO: 71;
an.antibody consisting ofaiheavy'chain.consisting of an.amino
acid sequence represented by amino acid numbers 20 to 471 in
SEQ ID NO: 85 and a light chain consisting of an amino acid
sequence ented by amino acid numbers 21 to 233 in SEQ
ID NO: 73;
an antibody consisting of a heavy chain.consisting of an amino
acid sequence represented by amino acid numbers 20 to 471 in
(followed by page 71a)
SEQ ID NO: 85 and a light chain consisting of an amino acid
sequence represented by amino acid numbers 21 to 233 in SEQ
ID NO: 75;
an antibody consisting of a heavy chain consisting of an amino
acid sequence represented by amino acid numbers 20 to 471 in
SEQ ID NO: 85 and a light chain consisting of an amino acid
ce represented by amino acid numbers 21 to 233 in SEQ
ID NO: 77;
an.antibody consisting of a heavy chain.consisting of an amino
acid sequence ented by amino acid numbers 20 to 471 in
SEQ ID NO: 85 and a light chain consisting of an amino acid
ce represented by amino acid numbers 21 to 233 in SEQ
ID NO: 79;
an dy consisting of a heavy chain consisting of an amino
acid sequence represented by amino acid numbers 20 to 471 in
SEQ ID NO: 85 and a light chain consisting of an amino acid
sequence represented by amino acid numbers 21 to 233 in SEQ
ID NO: 81;
an.antibody'consistingcfifa.heavy'chain.consisting of an amino
acid sequence represented by amino acid numbers 20 to 471 in
SEQ ID NO: 85 and a light chain consisting of an amino acid
sequence represented by amino acid numbers 21 to 233 in SEQ
ID NO: 83;
an antibody consisting of a heavy chain consisting of an amino
acid sequence represented by amino acid numbers 20 to 471 in
(followed by page 71b)
SEQ ID NO: 91 and a light chain consisting of an amino acid
sequence ented by amino acid numbers 21 to 233 in SEQ
ID NO: 71;
an dy ting of a heavy chain consisting of an amino
acid sequence represented by amino acid numbers 20 to 471 in
SEQ ID NO: 91 and a light chain consisting of an amino acid
sequence represented by amino acid numbers 21 to 233 in SEQ
ID NO: 73;
an.antibody consisting of a heavy chain.consisting of an.amino
acid sequence represented by amino acid numbers 20 to 471 in
SEQ ID NO: 91 and a light chain consisting of an amino acid
sequence represented by amino acid s 21 to 233 in SEQ
ID NO: 75; and
an antibody consisting of a heavy chain consisting of an amino
acid sequence represented by amino acid numbers 20 to 471 in
SEQ ID NO: 91 and a light chain consisting of an amino acid
sequence represented by amino acid numbers 21 to 233 in SEQ
ID NO: 77.
Furthermore, as an antibody' which has a. preferred
combination of a heavy chain and a light chain described above,
an antibody consisting of a heavy chain comprising an amino
acid sequence of SEQ ID NO: 85 and a light chain comprising
an amino acid ce of SEQ ID NO: 71; an antibody consisting
of a heavy chain comprising an amino acid sequence of SEQ ID
(followed by page 71c)
NO: 85 and a light chain comprising an amino acid sequence of
SEQ ID NO: 73; an antibody consisting of aa heavy chain
comprising an amino acid sequence of SEQ ID NO: 85 and.a light
chain comprising an amino acid sequence of SEQ ID NO: 75; an
antibody consisting of a heavy chain comprising an amino acid
sequence of SEQ ID NO: 85 and.a light chain comprising an.amino
acid ce of SEQ ID NO: 77; an antibody consisting of a
heavy chain sing an amino acid sequence of SEQ ID NO:
85 and a light chain
[FOLLOWED BY PAGE 72]
comprisingeniamino acid.sequence of SEQ IDDKR 79; an antibody
consisting of a heavy chain comprising an amino acid sequence
of SEQ ID NO: 85 and a light chain comprising an amino acid
sequence of SEQ ID NO: 81; an dy consisting of a heavy
chain comprising an amino acid sequence of SEQ ID NO: 85 and
a light chain comprising an amino acid sequence of SEQ ID NO:
83; an antibody consisting of a heavy chain comprising an amino
acid sequence of SEQ ID NO: 91 and a light chain comprising
an.amino acid sequence of SEQ IDINO: 71; an antibody ting
of a heavy chain sing an amino acid sequence of SEQ ID
NO: 91 and a light chain comprising an amino acid sequence of
SEQ ID NO: 73; an antibody consisting of ea heavy chain
comprising an amino acid sequence of SEQ ID NO: 91 and a light
chain comprising an amino acid sequence of SEQ ID NO: 75; and
an antibody consisting of a heavy chain comprising an amino
acid sequence of SEQ ID NO: 91 and a light chain comprising
an amino acid sequence of SEQ ID NO: 77 can be ified.
By combining a ce having a high homology with the
above-describedheavychainaminoacidsequencewitheasequence
having a high gy with the above—described light chain
aminoacidsequence,itispossibletoselectanantibodyhaving
a cytotoxic activity equivalent to that of each of the
above—described antibodies. Such a homology is generally a
homology of 80% or more, preferablgra homology of 90% orinore,
FP1214s 72 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
more preferably a homology of 95% or more, most preferably a
homology of 99% or more. Further, by combining an amino acid
sequence wherein one to several amino acid residues are
substituted,deletedcn:addedjxltheheavychaincn:lightchain
amino acid sequence, it is also possible to select an antibody
having a cytotoxic ty equivalent to that of each of the
above-described antibodies.
The homology n two amino acid sequences can be
determinedusingdefaultparameters(flfBlastalgorithmversion
2.2.2 (Altschul, Stephen F., Thomas L. Madden, dro A.
er, Jinghui Zhang, Zheng Zhang, Webb Miller, and David
J.Lipman(1997),“GappedBLASTandPSI—BLAST:anewgeneration
of protein database search programs”, Nucleic Acids Res. 25:
3389—3402). The Blast algorithm can also be used through the
Internet by accessing the site 1334907992153_O.
Incidentally, in the heavy chain amino acid sequence
representedbySEQIDNO:85,87,89<n:91intheSequenceListing,
the amino acid sequence consisting of amino acid residues 1
to 19 is a signal sequence, the amino acid.sequence consisting
of amino acid residues 20 to 141 is a variable region, and the
amino acid sequence consisting of amino acid residues 142 to
471 is a constant region.
Further, in the light chain amino acid sequence
s 73 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
represented by SEQ ID NO: 71, 73, 75, 77, 79, 81 or 83 in the
Sequence Listing, the amino acid sequence consisting of amino
acid residues 1 to 20 is a signal sequence, the amino acid
sequence consisting of amino acid residues 21 to 128 is a
variable region, and the amino acid sequence consisting of
amino acid es 129 to 233 is a constant region.
The heavy chain amino acid sequences represented by SEQ
ID NO: 85, 87, 89 or 91 in the Sequence Listing are encoded
by nucleotide sequences represented by SEQ ID NO: 84, 86, 88
or 90, respectively, in the Sequence Listing. Further, the
sequences represented by SEQ ID NOS: 84 and 85 are shown in
Fig. 34, the sequences represented by SEQ ID N08: 86 and 87
are shown in Fig. 35, the sequences represented.by SEQ ID NOS:
88 and 89 are shown in Fig. 36, and the sequences represented
by SEQ ID NOS: 90 and 91 are shown in Fig. 37. In each of the
above nucleotide sequences, the nucleotide sequence
tingofnucleotides1‘to57encodestheheavychainsignal
sequence of the dy, the nucleotide sequence consisting
ofnucleotides58ix)423encodestheheavychainvariableregion
of the antibody, and the nucleotide sequence consisting of
nucleotides424to1413encodestheheavychainconstantregion
of the dy.
The light chain amino acid sequences represented by SEQ
ID NO: 71, 73, 75, 77, 79, 81 or 83 in the ce Listing
are encoded by nucleotide sequences represented by SEQ ID NO:
FPl2l4s 74 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
70, 72, 74, 76, 78, 80 or 82, respectively, in the Sequence
g. Further, the ces represented by SEQ ID NOS:
70 and 71 are shown in Fig. 27, the sequences represented by
SEQ ID NOS: 72 and 73 are shown in Fig. 28, the sequences
represented by SEQ ID NOS: 74 and 75 are shown in Fig. 29, the
sequences represented by SEQ ID NOS: 76 and 77 are shown in
Fig. 30, the sequences represented by SEQ ID NOS: 78 and 79
are shown in Fig. 31, the sequences ented by SEQ ID NOS:
80 and 81 are shown in Fig. 32, and the sequences represented
by SEQ ID NOS: 82 and 83 are shown in Fig. 33. In each of the
above nucleotide sequences, the tide sequence
consistingofrnxfleotides111360encodesthelightchainsignal
sequence of the antibody, the nucleotide sequence ting
ofnucleotides61113384encodesthelightchainvariableregion
of the antibody, and the nucleotide sequence consisting of
nucleotides 385 to 699 encodes the light chain constant region
of the antibody.
The homology between any of these nucleotide sequences
and a nucleotide sequence of another antibody can also be
determined using the Blast algorithm.
r, the antibody of the invention includes a human
antibody which binds to the same epitope as the M30 antibody.
An.anti—B7—H3 human antibody refers to a human antibody having
FP1214s 75 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
only a gene sequence of an antibody derived from a human
chromosome. The anti—B7-H3 human antibody can be obtained by
a method usingzahuman.antibody—producing1nouseihavingeahuman
chromosome fragment comprising heavy and light chain genes of
a human antibody (see Tomizuka, K. et al., Nature Genetics
(1997) 16, pp. 133—143; Kuroiwa, Y. et al., Nucl. Acids Res.
(1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell
Technology: Basic and Applied Aspects vol. 10, pp. 69—73
(Kitagawa,Y.,Matuda,T.andIijima,S.eds.),KluwerAcademic
Publishers, 1999; ka, K. et al., Proc. Natl. Acad. Sci.
USA (2000) 97, pp. 722-727, etc.).
Such a human antibody—producing mouse can be created
specifically as follows. A genetically modified animal in
whichendogenousimmunoglobulinheavyandlightchaingeneloci
have been disrupted, and instead, human globulin heavy
and light chain gene loci have been introduced via a yeast
artificial chromosome (YAC) vector or the like is created by
producing a knockout animal and a transgenic animal andInating
these animals.
Further, according to a recombinant DNA que, by
using cDNAs encoding each of such a heavy chain and a light
chain of a human antibody, and preferably a vector comprising
such cDNAs, eukaryotic cells are ormed, and a
FP1214s 76 810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
transformant cell which produces a recombinant human
monoclonal antibody is ed, whereby the dy can also
be obtained from the culture supernatant.
Here, as the host, for example, eukaryotic cells,
preferablylnammalian cells such as CHO cells, lymphocytes, or
myeloma cells can be used.
Further, a method of obtaining a phage display-derived
human antibody ed from a human antibody library (see
Wormstone, I. M. et al., Investigative Ophthalmology & Visual
Science. (2002) 43 (7), pp. 2301—2308; Carmen, S. et al.,
Briefings in Functional Genomics and Proteomics (2002), 1 (2),
pp. 189—203; rdena, D. et al., Ophthalmology (2002) 109
(3), pp. 427-431, etc.) is also known.
For example, a phage display method in which.a variable
region of a human antibody is expressed on the surface of a
phageasaasingle—chainantibody(scFv),andeaphagewhichbinds
to an antigen is selected (Nature Biotechnology (2005), 23,
(9), pp. 1105—1116) can be used.
By analyzing the gene of the phage selected based on its
binding to an antigen, a DNA sequence encoding the le
region of a human antibody which binds to an antigen can be
FP1214s 77 WGA/PN810023/Bng trans of PCT spec/26.9.13
4426167—1—WARENDS
determined.
If the DNA sequence of scFv which binds to an antigen
is determined, a human antibody can be ed by preparing
an expression vector comprising the sequence and introducing
the vector intt>an.appropriate host to express it (WO 92/01047,
WC)92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WC>95/01438,
WC>95/15388, Annu. Rev: Immunol. (1994) 12, pp. 433-455, Nature
Biotechnology (2005) 23 (9), pp. 1105—1116).
If a newly produced human antibody binds to a partial
peptide or a partial ry structure to which the M30
antibody binds, it can be determined that the human antibody
binds to the same epitope as the M30 antibody. Further, by
confirming that the human antibody es with the M30
antibody for binding to B7-H3 (that is, the human antibody
ts the binding between the M30 antibody and B7—H3), it
can be determined that the human antibody binds to the same
epitope as the M30 antibody even if the specific epitope
sequence or structure has not been determined. When it is
confirmed that the human antibody binds to the same epitope
as the M30 antibody, the human antibody is strongly expected
to have a cytotoxic activity equivalent to that of the M30
antibody.
{0128]
s 78 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
The chimeric dies, humanized.antibodies, or human
antibodies obtained by the above-described methods are
evaluated for their n—binding properties by a method
shown in Example 3 or the like, and a preferred antibody can
be selected.
As one example of another index for use in the comparison
of the properties of antibodies, the stability of antibodies
cankxaexemplified. .Adifferentialscanningcalorimeter(DSC)
is a device capable of quickly and accurately measuring a
thermal ration midpoint temperature (Tm) to be used as
a ble index of the relative conformational stability of
proteins . By ing the Tm values using a DSC and comparing
the values, a difference in thermal stability can.be compared.
It is known that the storage stabilit37of'antibodies shows some
correlation with the thermal stability of antibodies (Lori
Burton, et. al., Pharmaceutical Development and Technology
(2007) 12, pp. 265—273), and a preferred antibody can be
selected by using l stability as an index. Examples of
other indices for selecting antibodies include the following
features: the yield in an appropriate host cell is high; and
the aggregability:h1an aqueous solution.is low. For example,
an antibody which shows the highest yield does not always show
the highest thermal stability, and therefore, it is necessary
to select an dy most suitable for the administration to
FP1214s '79 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
humans by making comprehensive evaluation based on the
above—described indices.
Further, a method in which the full—length heavy and
light chain sequences of an antibody are connected using an
appropriate linker, whereby a single—chain immunoglobulin is
obtainedisalsoknown(Lee,H—S,et.al.,MolecularImmunology
(1999) 36, pp. 61—71; Shirrmann, T. et. al., mAbs (2010), 2,
(1) pp. 1—4). By dimerizing such a single-chain
globulin, the ing dimer can have a structure and
an activity similar to those of an antibody which is a tetramer
. Further, the antibody of the invention may be an
antibody which has a single heavy chain variable region and
does not have a light chain sequence. Such an antibody is
called a single domain dy (sdAb) or a nanobody, and in
fact, such an dy is observed in camels and llamas and
has been reported to have an antigen-binding affinity
(Muyldemans S. et. al., Protein Eng. (1994) 7 (9), 1129~35,
Hamers—Casterman C. et. al., Nature (1993) 363 (6428) 446—8).
The above-described.antibodies can e construed aseatype
of functional fragment of the dy according to the
invention.
In the invention, a modified variant of the antibody or
a functional fragment of the antibody is also included. The
modified variant refers to a variant obtained by subjecting
FP1214s 8O WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
the antibody or a functional fragment of the antibody of the
ion to chemical or biological modification. Examples
of such a chemically modified variant include variants
chemically modified by linking a chemical moiety to an amino
acid skeleton, variants chemically modified with an N—linked
or O—linked carbohydrate chain, etc. es of such a
biologically modified variant include ts obtained by
modification after translation (such as N—linked or O—linked
glycosylation, DP- or C—terminal processing, deamidation,
isomerization of aspartic acid, or oxidation of methionine),
and variants in which a methionine residue has been added to
the N terminus by being expressed in a prokaryotic host cell.
Further, an antibody labeled so as to enable the
detection or isolation of the antibody or an antigen of the
invention, for example, an enzyme-labeled antibody, a
fluorescence-labeled antibody, and an affinity—labeled
antibody are also included in the g of the modified
t. Such a nwdified variant of the antibody or a
functional fragment of the antibody of the invention is useful
for ing the stability and blood retention of the original
antibody or a functional fragment of the antibody of the
invention, reducing the antigenicity thereof, detecting or
isolating such an antibody or an antigen, and so on.
Further,byregulatingthenmdificationofkaglycanwhich
is linked to the antibody of the invention (glycosylation,
FP1214s 81 WGA/PN810023/Eng trans of PCT 6.9.13
4426167WARENDS
defucosylation, etc.), it is possible to
enhanceantibody—dependent cellular cytotoxic activity. As
the technique for regulating the modification of a glycan of
antibodies, WO 99/54342, WO 00/61739, WO 02/31140, etc.
disclose known ques. However, the technique is not
d.thereto. In the antibody and the onal fragment
of the antibody of the invention, an antibody or a functional
fragment of the dy in which.the modification of a glycan
is regulated is also included.
Incaseswhereaulantibodyjjsproducedknlfirstisolating
an antibody gene and then introducing the gene into an
appropriate host, a combination of an appropriate host and an
appropriate expression vector can.be used. Specific examples
of the antibody gene e a combination of a gene encoding
a heavy chain sequence of an antibody and a gene encoding a
light chain.sequence thereof described.in this ication.
When a host cell is transformed, it is possible to insert the
heavy chain sequence gene and the light chain ce gene
into the same expression vector, and also into different
expression vectors separately.
In cases where eukaryotic cells are used as the host,
animal cells, plant cells, and eukaryotic microorganisms can
be used. As such animal cells, mammalian cells, for example,
simian COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175—182,
FP1214s 82 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
ATCC GEL-1650) NIH3T3
, murine lasts (ATCC NO. GEL-1658) ,
and<dihydrofolate ase—deficient strains (Urlaub, G. and
Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, pp.
4126-4220) of Chinese hamster ovarian cells (CHO cells; ATCC:
CCL—6l) can be exemplified.
In cases where prokaryotic cells are used, for example,
Escherichia coli and Bacillus subtilis can be ified.
By introducing a gene of 21 desired antibody or a
functional fragment of the antibody into these cells through
transformation, and culturing the thus ormed cells in
vitro, the antibody can be obtained. In the above—described
culture method, the yield may sometimes vary depending on the
sequence of the antibody, and therefore, it is le to
select an antibody which is easily produced as a pharmaceutical
by using the yield as an index among the antibodies having an
equivalent binding activity. Therefore, in the antibody and
the functional fragment of the antibody of the invention, an
antibody or a functional nt of the antibody obtained by
a method.of producing an antibody or a onal fragment of
the antibody, characterized by including a step of culturing
the transformed host cell and a step of collecting a desired
antibody or a onal fragment of the antibody from a
cultured product obtained in the culturing step is also
included.
Incidentally, it is known that a lysine residue at the
carboxyl terminus of the heavy chain of an antibody produced
in a cultured mammalian cell is deleted (Journal of
Chromatography A, 705: 129—134 (1995)), and it is also known
that two amino acid residues (glycine and lysine) at the
carboxyl terminus of the heavy chain of an antibody produced
inaacultureddnammalian cell are deleted.andeaproline residue
newlylocatedafl:thecarboxylterminusjjsamidated(Analytical
Biochemistry, 360: 75—83 (2007)). However, such deletion and
modification of the heavy chain sequence do not affect the
antigen—binding ty and the effector function (the
activation of complement, the antibody—dependent cellular
cytotoxicity, etc.) of the antibody. Therefore, in the
invention, an antibody and a functional fragment of the
antibody subjectedixDsuchanodification.are also included, and
a deletion t in which one or two amino acids have been
d at the carboxyl terminus of the heavy chain, a variant
ed by amidation of the on variant (for e,
a heavy chain in which the carboxyl terminal proline residue
has been amidated), and the like can be exemplified. The type
of deletion variant having'a<ieletion.at the yl terminus
of the heavy chain of the antibody according to the invention
is not limited to the above variants as long as the
antigen—binding affinity and the effector function are
conserved. The two heavy chains constituting the antibody
according to the invention may be of one type selected from
FPl2l4s 84 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1~WARENDS
the group consisting of a full—length heavy chain and the
above—described deletion variant, or may be of two types in
combination selected therefrom. The ratio of the amount of
each deletion variant can be ed by the type of cultured
mammalian cells which produce the antibody according to the
invention and the culture conditions, however, a case where
noacidresidueafl:thecarboxylterminushasbeendeleted
in both of the two heavy chains contained as main components
in.the dy according to the invention cankxaexemplified.
There is no limitation on isotype of the antibody of the
ion,andexamplesthereofincludeIgG(IgGl,IgG2,lgG3,
IgG4), IgM, IgA (IgAl, IgA2), IgD, and IgE, and preferred
examples thereof include IgG and IgM, and further' more
preferred examples thereof include IgGl and IgG2.
Further, the antibody of the invention may be a
functional fragment of the antibody having an antigen—binding
site of the antibody or a modified fragment thereof. The
fragment of the antibody can be obtained by treating the
antibody with a protease such as papain or pepsin, or modifying
the antibody’gene ing toaagenetic:engineering technique
and expressing the ed gene in suitable cultured cells.
Among these antibody fragments, a fragment having all or part
of the functions of the dy can be called a functional
fragment of the antibody.
As the functions of the antibody, generally
FP1214s 85 WGA/PN810023/Eng trans of PCT 6.9.13
4426167~1~WARENDS
antigen—binding ty, an ty of neutralizing the
activity of an antigen, an activity of enhancing the activity
of an antigen, antibody—dependent cellular cytotoxicity
(ADCC) activity and complement-dependent cytotoxicity (CDC)
activity can be exemplified. The function of the antibody and
the functional fragment of the antibody according to the
invention is a binding activity to B7—H3, preferably
antibody—dependent cell—mediated ytosis (ADCP)
activity, more preferably cytotoxic activity (antitumor
activity) mediated by an ADCP activity against tumor cells.
Further, the antibody of the invention.may have ADCC activity
and/or CDC ty in dn to .ADCP ty. In
particular, it has been reported that a pharmaceutical
containing a currently available antitumor antibody directly
acts on tumor cells to block a proliferative signal, directly
acts on tumor cells to induce a cell death signal, suppresses
angiogenesis, s ADCC ty1nediated.by NK cells, and
induces CDC activity mediated by complement, thereby
suppressing the growth of tumor cells (J Clin Oncol 28:
4390—4399. (2010), Clin Cancer Res; 16 (1); 11-20. (2010)),
however, at least the present inventors are not aware that the
ADCP activity of the anti—B7—H3 antibody according to the
inventimiofthisapplicationhasbeenreportedastheactivity
ofaapharmaceuticalcontainingaacurrentlyavailableantitumor
antibody.
FP1214s 86 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1~WARENDS
Examples of the fragment of the antibody include Fab,
F(ab’)2, Fv, single—chain Fv (scFv) in which Fv molecules of
the heavy chain and the light chain are connected via an
appropriate y a diabody (diabodies), a linear antibody,
and a polyspecific antibody composed of the antibody fragment.
Further, Fab' which is a monovalent nt in a variable
region of an antibody obtained by treating F(ab’)2 under
ng ions is also included in the fragment of the
antibody.
Further, the antibody of the invention may be a
polyspecific antibody‘ with specificity for' at least two
ent types of antigens. In general, such an antibody
binds to two types of antigens (that is , a bispecific antibody) ,
however, the “polyspecific antibody” as used.herein includes
an dy having specificity for two or more (for example,
three) types of antigens.
The polyspecific antibody of the invention may be a
full—length antibody or a fragment of such an antibody (for
example, a F(ab’)2 bispecific antibody). The bispecific
antibody can be produced by connecting the heavy and light
chains (HL pairs) of two types of antibodies, or can also be
produced by fusing hybridomas which produce different
onal antibodies to prepare bispecific
antibody—producing fused cells (Millstein et al., Nature
(1983) 305, pp. 537—539).
The antibody of the invention may be a single—chain
antibody (also referred. to as scFv). The single—chain
antibody can be obtained by connecting the heavy chain variable
region and the light chain.variable region of the antibody via
a polypeptide linker (Pluckthun, The Pharmacology of
Monoclonal Antibodies, 113 (edited by Rosenberg and Moore),
Springer Verlag, New York, pp. 5 (1994), Nature
Biotechnology (2005), 23, pp. 1126-1136). Further, a BiscFv
fragment produced by ting two scFv les via a
polypeptidelinkercanalsobetwedansthebispecificantibody.
A method of producing a single—chain dy is known
in this technical field (see, for example, US patent Nos.
4,946,778, 203, 5,091,513, 5,455,030, etc.). In this
scFv, the heavy chain variable region and the light chain
variable region are connected.via a linker which does not form
a conjugate, preferably via a polypeptide linker (Huston, J.
S. et al., Proc. Natl. Acadd Sci. USA.(1988), 85, pp. 5879—5883).
InthescFv,theheavychainvariableregionandthelightchain
variable region may be derived from the same antibody or
different antibodies.
FP1214s 88 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
As the polypeptide linker to be used for connecting the
variable regions, for example, a given single—chain peptide
consisting of 12 to 19 residues is used.
A DNA encoding an scFv can be obtained by performing
amplification through PCR using a DNA as a template that
comprises all or desired part of a DNA selected from a DNA
encoding the heavy chain or the heavy chain variable region
of the above—described antibody and a DNA encoding the light
chairlor the light chain variable ‘thereof'and‘also using
a primer pair that defines both ends of the template DNA, and
further performing amplification by combining a DNA encoding
a polypeptide linker portion and a primer pair that defines
both ends of the polypeptide so as to connect said both ends
f to each of the heavy chain and the light chain.
Further, once DNA encoding an scFv is ed, an
expression vector comprising the same and a host transformed
by the expression vector can be obtained according to common
ures. Further, by using the resulting host, an scFV'can
be ed according to common procedures. An dy
fragment thereof can be produced in a host by obtaining a gene
and expressing the gene in the same manner as described above.
The antibody of the invention may be multimerized to
FP1214S 89 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
increase its affinity for an antigen. The antibody to be
multimerized may be one type of antibody or plural antibodies
which recognize plural epitopes of the same antigen. As a
method.of multimerization of the antibody, binding of the IgG
CH3 domain to two scFv molecules, binding to streptavidin,
uction of'a helix—turn—helix motif, and the like can be
ified.
The antibody of the ion may be ea polyclonal
antibody which is a mixture of plural types of anti-B7—H3
antibodies having different amino acid sequences. As one
example of the polyclonal antibody, a mixture of plural types
ofantibodieshavingdifferentCDchnibeexemplified. Assuch
a polyclonal antibody, antibodies obtained by culturing a
mixture of cells which produce different antibodies and then
purifying the antibodies fronlthe resulting culture used
(see ).
As a modified antibody, an antibody bound to any of
various types of molecules such as hylene glycol (PEG)
can also be used.
Further, the dy of the iorlmagrbe in the form
of a conjugate formed between any of these antibodies and
another medicinal agent (immunoconjugate). Examples of such
FP1214S 90 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
an antibody include a conjugate in which the antibody is
conjugated to a radioactive material or a compound having a
pharmacological action (Nature Biotechnology (2005) 23, pp.
1137—1146). Examples thereof include indium (“lIn)-capromab
pendetide, technetium (9%Tc)-nofetumomab merpentan, indium
(lnIn)—ibritumomab, yttrium BOY)-ibritumomab, and iodine
(1“I)-tositumomab.
The ed antibody can be purified to homogeneity.
The separation and purification of the antibody may be
performed employing a conventional protein separation and
purification method. For example, the antibody can be
separated and ed by appropriately selecting and
combining column chromatography, filter tion,
iltration, salt itation, dialysis, preparative
polyacrylamide gel electrophoresis, isoelectric focusing
electrophoresis, and the like (Strategies for Protein
Purification and.Characterization: AILaboratory'Courselflanual,
Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory
Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and
David Lane, Cold Spring Harbor tory (1988)), but the
method is not limited thereto.
Examples of such chromatography e affinity
chromatography, ion exchange chromatography, hydrophobic
FP1214S 91 WGA/PN810023/Eng trans of PCT 6.9.13
4426167~1-WARENDS
chromatography, gel filtration chromatography, reverse phase
chromatography, and adsorption chromatography.
Such chromatography can be performed employing liquid
chromatography such as HPLC or FPLC.
As a column to be used in affinity chromatography, a
n A column and a Protein G column can be exemplified.
For example, as a column using a Protein A column, Hyper D,
POROS, Sepharose FF (Pharmacia) and the like can be
exemplified.
Further,byusingwacarrierhavingenlantigenimmobilized
thereon, the antibody can also be purified utilizing the
binding ty of the antibody to the antigen.
3. Pharmaceutical comprising anti—B7-H3 antibody
The antibodies obtained by the method bed in the
above item “2. Production of antieB7—H3 antibody” exhibit a
cytotoxic activity against cancer cells, and ore can be
used as a pharmaceutical, particularly a therapeutic agent
and/or preventive agent for cancer.
Thecytotoxic activity exhibited.by an antibody in vitro
can be determined by measuring the inhibitory activity of cell
growth.
FP1214S 92 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
Forexample,aacancercelllinewhichoverexpressesB7—H3
is cultured, an antibody is added to the culture system at
different concentrations, and any inhibitory activity against
focus formation, colony formation, and spheroid growth can be
measured.
The in vivo therapeutic effect of an dy on cancer
using experimental s can be determined by, for example,
administering the antibody to nude mice implanted with a tumor
cell line which overexpresses B7—H3 and measuring any change
in the cancer cells.
Examples of the type of cancer include lung ,
kidney cancer, urothelial carcinoma, colorectal cancer,
prostate cancer, glioblastoma multiforme, ovarian cancer,
atic cancer, breast cancer, a melanoma, liver cancer,
bladdercancer,stomachcancer,andesophagealcancer,however,
the type of cancer is not limited.thereto as long as the cancer
cell to be treated expresses B7—H3.
An acceptable nce to be used in the preparation
of the pharmaceutical composition according to the invention
is preferably non—toxic to a individual to whom the
pharmaceutical composition is to be administered in terms of
the dose and tration.
FPlZl4s 93 WGA/PN8lOOZ3/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
The pharmaceutical composition of the ion can
comprise a substance for pharmaceutical use which is capable
ofchangingornwintainingthepH,osmoticpressure,Viscosity,
transparency, color, isotonicity, aseptic condition,
stability, solubility, release rate, absorption rate, and
permeability’ thereof. Examples of such. a substance for
pharmaceuticaluseinclude,butarenotlimitedto,aminoacids
sudnasglycine,alanine,glutamine,asparagine,arginine,and
lysine; antimicrobial agents; antioxidants such as ascorbic
acid,sodiumsulfate,andsodiumhydrogensulfite;bufferssuch
as phosphate, e, borate buffers, sodium hydrogen
carbonate, and Tris—HCl solutions; fillers such as mannitol
and glycine; ing agents such as ethylenediamine
tetraacetate (EDTA); complexing agents such as caffeine,
polyvinylpyrrolidine, B—cyclodextrin, and
hydroxypropyl—B—cyclodextrin; expanders such as glucose,
e, and n; other carbohydrates such as
monosaccharides and disaccharides; coloring agents; flavors;
diluents; emulsifying agents; hydrophilic polymers such as
polyvinylpyrrolidine; preservatives such as low—molecular
weight polypeptides, salt—forming r ions, benzalkonium
chloride, benzoic:acid, salicylic acid, osal, phenethyl
l, methylparaben, propylparaben, chlorhexidine, sorbic
acid, and. hydrogen. peroxide; solvents such. as glycerin,
FP1214s 94 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167~1~WARENDS
propyleneglycol,andpolyethyleneglycol;sugaralcoholssuch
asxnannitol and sorbitol; suspending agents; surfactants such
as sorbitan ester, polysorbates including polysorbate 20 and
polysorbate 80, Triton, tromethamine, lecithin, and
cholesterol; stability enhancing agents such as sucrose and
sorbitol; elasticity?enhancingwagents sucflas sodiuntchloride,
potassium chloride, and ol and sorbitol; transport
agents; excipients; and/or pharmaceutical adjuvants. The
ofthesesubstancesforpharmaceuticaluseispmeferably
from 0.001 to 100 times, particularly ably from 0.1 to
times the weight.of the anti—B7-H3 antibody. Those skilled
irlthe art can appropriately determine a1oreferred formulation
of the pharmaceutical composition in a preparation depending
on the e to which the composition is applied, the route
of administration to be d, or the like.
The excipient or carrier in the pharmaceutical
composition may be in the form of a liquid or a solid. An
appropriate excipient or carrier may be injectable water,
logical saline, an artificial cerebrospinal fluid, or
other substance commonly used for parenteral administration.
r, neutral physiological saline or physiological saline
containing serum albumin can also be used as a carrier. The
pharmaceutical composition may contain a Tris buffer of pH 7 . O
to 8.5, an acetate buffer of pH 4.0 to 5.5, or‘a citrate buffer
FP1214S 95 810023/Eng trans of PCT speC/26.9.l3
4426167—1-WARENDS
of pH 3.0 to 6.2. Further, such a buffer may be supplemented
with sorbitol or another compound.
es of the pharmaceutical composition. of the
inventionincludeeapharmaceuticalcompositioncnmprisingthe
anti-B7—H3 antibody and a pharmaceutical composition
comprisingtfluaanti—B7—H3antibodyammiatleastcxmatherapeutic
agent for cancer. The pharmaceutical composition of the
invention is prepared in the f a lyophilized product or
a liquid as a medicinal agent having a selected composition
and a required purity. The ceutical composition
sing the anti—B7—H3 antibody and the ceutical
composition comprising the anti—B7—H3 antibody and at least
one therapeutic agent for cancer can also be formed into a
lyophilized product using an appropriate excipient such as
sucrose.
In the above—described pharmaceutical composition, the
therapeutic agent for cancer to be incorporated along with the
anti—B7—H3 antibody may be administered simultaneously with,
telyfromy(n:sequentiallyvfiifi1theanti—B7—H3antibody,
or the therapeutic agent and the anti—B7—H3 antibody may be
administered at different dosage intervals. Examples of such
a therapeutic agent for cancer include abraxane, carboplatin,
cisplatin, abine, irinotecan (CPT-ll), paclitaxel,
FP1214S 96 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
pemetrexed, nib, vinblastin, and medicinal agents
described in , and additional examples thereof
include LH—RH analogues (such as leuprorelin and goserelin),
estramustine phosphate, estrogen antagonists (such as
tamoxifen and raloxifene), and aromatase inhibitors (such as
anastrozole, letrozole, and exemestane), however, the agent
is not limited thereto as long as the agent is alnedicinal agent
having an antitumor activity.
An individual to whontthe pharmaceutical composition is
to be administered is not particularly limited, however,
preferred are mammals, and more preferred are humans.
The pharmaceutical composition of the invention can be
prepared for parenteral stration or for
intestinalabsorptionthroughoraladministration. The
composition and concentration of a preparation can be
determineddepending(Hitheadministrationnethod. Thehigher
the affinity of the anti—B7~H3 antibody comprised in the
pharmaceutical.composition of the invention is for B7-H3, that
is, the lower the iation constant (Kd value) thereof is
for B7—H3, the more the 7—H3 antibody can exhibit its
drug efficacy even when decreasing the dose for humans . Hence,
the dose of the ceutical composition of the invention
for humans can also be determined based on this result. As
for the dose, in the case where a human anti—B7—H3 antibody
S 97 WGA/PN810023/Eng trans of PCT speC/26.9.l3
4426167WARENDS
is administered to , the antibody may be administered
ataadose of fronlabout 0.001 to 100 mg/kg once or l times
at intervals of 1 to 180 days. Examples of the dosage form
of the pharmaceutical composition of the invention include
ions including infusions, suppositories, transnasal
agents, sublingual agents, and percutaneous absorbents.
Examples
Hereinafter, the invention will be described in more
detail in Examples, however, the invention is not limited
thereto.
Note that the respective operations regarding gene
(followed by page 99)
manipulation in the following Examples were performed
according to the methods described in "Molecular g"
(written by ok, J., Fritsch, E. F. and Maniatis, T.,
hed by Cold Spring Harbor Laboratory Press in 1989), or
in the case of using commercially available reagents or kits,
they are used according to the instructions attached thereto
unless ise stated.
Example 1. Production of plasmid
l)—l Production of human B7-H3 expression vector
l)-l—l Production of expression vector for full—length human
B7-H3 variant 1
A PCR reaction was performed using a cDNA synthesized
from the total RNA of LNCaP cells (American Type Culture
tion (ATCC)) as a template and also using the following
primer set, thereby amplifying a cDNA encoding the human B7~H3
variant 1:
Primer l:
’—ctatagggagacccaagctggctagcatgctgcgtcggcggggcag—3’ (SEQ
ID NO: 1 in the Sequence Listing); and
Primer 2:
’—aacgggccctctagactcgagcggccgctcaggctatttcttgtccatcatcttc
tttgctgtcag-3' (SEQ ID NO: 2 in the Sequence Listing).
Subsequently,thethusobtainedPCRproductwaspurified
usingMagEXtractorPCRékGelcleanup(TOYOBO,Co.,Ltd.). Then,
the PCR product was digested with restriction enzymes (Nhel
and NotI), followed by purification using MagExtractor PCR &
Gel cleanup (TOYOBO, Co., Ltd.). A pcDNA3.l(+) plasmid DNA
wasdigestedwiththesamerestrictionenzymes(NheIandNotI),
followed by purification using MagExtractor PCR &<3el cleanup
(TOYOBO, CO. , Ltd.) .
The ing purified DNA solutions were mixed, and
further, Ligation high (TOYOBO, Co., Ltd.) was added o,
and the resulting mixture was incubated at 16°C for 8 hours
to effect ligation. The resulting reaction.mixture was added
to E. coli DHSd competent cells (Invitrogen Corporation) to
effect transformation.
ColonydirectPCRwasperformedcnitheresultingcolonies
using the PCR primers and the BGH reverse primer, and a
candidate clone was selected.
The obtained candidate clone was cultured in a liquid
medium (TB/Amp), and 51 plasmid IMWX was extracted using
MagExtractor ~Plasmid— (TOYOBO, Co., Ltd.).
By using the obtained plasmid DNA as a te, a
sequence between the following Primer 3 and Primer 4 was
determinadbysequenceanalysisandthesequenceswerecompared
between the obtained clone and the ed CDS sequence:
Primer 3 (CMV promoter primer):
’—cgcaaatgggcggtaggcgtg-3’ (SEQ ID NO: 3 in the Sequence
Listing); and
S 100 810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
Primer 4 (BGH reverse primer):
'-tagaaggcacagtcgagg-3’ (SEQ ID NO: 4 in the Sequence
Listing).
After confirming the sequence, the obtained clone was
cultured in 200 ml of LB/Amp medium, and a plasmid DNA was
extracted using Plasmid Midi V—lOO kit ne, Inc.).
The thus obtained vector was named “pcDNA3.l—B7-H3”.
ThesequenceoftflmaORFrengioftheB7—H3variant]_genecloned
in this vector is represented by nucleotide numbers 1 to 1602
in SEQ ID NO: 5 in the Sequence Listing. Further, the amino
acid sequence of the B7—H3 variant 1 is represented by SEQ ID
NO: 6 in the Sequence Listing.
l)—l—2 Production of expression vector for full—length human
B7—H3 variant 2
PCR was med using a cDNA synthesized from the total
RNA of LNCaP cells as a template and also using the ing
primer set, thereby amplifying a cDNA.encoding the human B7—H3
variant 2:
Primer 5
’~ggggacaagtttgtacaaaaaagcaggcttcaccatgctgcgtcggcggggcagc
cctg—3’ (SEQ ID NO: 7 in the Sequence g)
Primer 6
’-ggggaccactttgtacaagaaagctgggtcggctatttcttgt-3’ (SEQ ID
NO: 8 in the Sequence g).
FP1214s 101 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
Purification was performed in the same manner as in
Example l)—l-l, and the PCR product after purification was
ated.intoaapDONRZZl vector (Invitrogen Corporation) by
a Gateway BP reaction, thereby transforming E. coli TOPlO
(Invitrogen Corporation).
For the clones obtained after transformation, the size
of the insert was confirmed by colony PCR. For 8 clones in
which the size of the insert was confirmed, the DNA sequence
at the 3’ end and the 5’ end of the insert was confirmed by
performing one sequencing reaction fronlthe vector side to the
insert side for both ends. A Gateway LR reaction between the
entry clone whose sequence was med and a Gateway
destination vector pcDNA—DEST4O (Invitrogen Corporation) was
performed. For the clone obtained after transformation of E.
coli.TOP10, the size of the insert was confirmedkn/colony PCR.
For the clone in which the size of the insert was confirmed,
the DNA sequence at the 3’ end and the 5’ end of the insert
was analyzed to confirm that the insert of interest was
correctlyinserted. tlxmgofthethusproducedplasmid
of the clone was purified using PureLink HiPure Plasmid
Megaprep Kit (Invitrogen Corporation).
The thus obtained vector was named “pcDNA—DEST40—B7—H3
t 2” . The sequence of the ORF region of the B7—H3 variant
2 gene cloned in this vector is ented by nucleotide
s 102 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
numbers 1 to 948 in SEQ ID NO: 9 in the ce Listing.
Further, the amino acid sequence of the B7—H3 variant 2 is
represented by SEQ ID NO: 10 in the Sequence Listing.
1)—2 tion of expression vector for B7—H3 partial.protein
By using the B7—H3 full—length plasmid related to the
B7-H3 variant 1 of Example 1)—1~1 as a template, each of the
following regions was amplified by PCR. The numbers g
each region of interest pond to the nucleotide numbers
of B7—H3 represented.by SEQ ID NO: 5. The primer was designed
to contair1a stop codon at the 3’ end.in addition to the Gateway
att sequence.
Each of the following regions 1), 2), and 3) was prepared
by ying two regions and.then ligating the regions by PCR
to ne fragment. That is, as for region 1), amplification
was performed using Primers 7 and 12, and Primers 15 and 11,
and the resulting PCR products were further amplified using
Primers'7and11. .AsforregionZ),amplificationwasperformed
using Primers 8(and 13, and.Primers 15 and 11, and the resulting
PCR products were further amplified using Primers 8 and 11.
As for region 3), amplification was performed using s
9 and 14, and Primers 15 and 11, and the resulting PCR products
were further amplified using Primers 9 and 11. As for region
4), amplification was performed using Primers 10 and 11. As
for region 5), amplification was performed using Primers 8(and
FP1214S 103WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
11. As for region 6), amplification was performed using
Primers 9 and 11.
Regions of interest
1) B7—H3 variant 1 ORF: 79—417 and 1369—1602 (573 bp)
2) B7-H3 variant 1 ORF: 418—732 and 1369—1602 (549 bp)
3) B7—H3 variant 1 ORF: 733—1071 and 1369—1602 (573 bp)
4) B7—H3 t 1 ORF: 1072~1602 (531 bp)
) B7—H3 t 1 ORF: 418—1602 (1185 bp)
6) B7—H3 variant 1 ORF: 733~1602 (870 bp)
Primer number and base sequence
Primer 7
’—ggggacaagtttgtacaaaaaagcaggcttcggagccctggaggtccaggtc—3’
(SEQ ID NO: 11 in the Sequence Listing)
Primer 8
’—ggggacaagtttgtacaaaaaagcaggcttcgctccctactcgaagcccagcatg
—3’ (SEQ ID NO: 12 in the Sequence Listing)
Primer 9
’—ggggacaagtttgtacaaaaaagcaggcttcggagccgtggaggtccaggtc—3’
(SEQ ID NO: 13 in the ce Listing)
Primer 10
’~ggggacaagtttgtacaaaaaagcaggcttcgctccctactcgaagcccagcatg
—3’ (SEQ ID NO: 14 in the Sequence Listing)
Primer 11
FP12145 104 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
’—ggggaccactttgtacaagaaagctgggtctcaggctatttcttgtccatcatc—
3’ (SEQ ID NO: 15 in the Sequence Listing)
Primer 12
’—gggaatgtcataggctgcccggccacctgcaggctgacggcag—3’ (SEQ ID
NO: 16 in the Sequence Listing)
Primer 13
’—gggaatgtcataggctgccctgtggggcttctctggggtgtg—3’ (SEQ UDNO:
17 in the Sequence Listing)
Primer 14
'—gggaatgtcataggctgcccggccacctgcaggctgacggcag—3’ (SEQ ID
NO: 18 in the ce Listing)
Primer 15
’—gggcagcctatgacattccccccagag—3’ (SEQ ID NO: 19 in the
ce Listing)
Purification was performed in the same manner as in
Example 1)—1—1, and each of the amplified products after
purificationwasintegratedintoagflkflRZZlvectorbywaGateway
BP reaction, thereby transforming E. coli TOPlO. For the
clones obtained after transformation, the size of the insert
was confirmed by colony PCR.
For each of the clones in which the size of the insert
was confirmed, the DNA sequence at the 3’ end and the 5’ end
of the insert was confirmed by performing one sequencing
reaction fronlthe vector side to the insert side for both ends.
For the clones which were confirmed to have the insert
S 105 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
of interest, the total DNA ce of the insert was also
confirmedusingthefellowingprimers. Asaaresultofsequence
is, it was confirmed that all of the sequences were
completely identical to the information of the sequences of
interest.
A Gateway LR reaction between each of the entry clones
whose sequence was confirmed and pFLAG—myc—CMV—l9—DEST
(Invitrogen Corporation) was med. For the clones
obtained after transformation of E. coli DHlOB (Invitrogen
Corporation), the size of the insert was confirmed by colony
PCR.
For each of the clones in which the size of the insert
was confirmed, the DNA sequence at the 3’ end and the 5' end
of the insert was analyzed to confirm that the insert of
interest was correctly ed. Hereinaftery the expression
vectors obtained by integrating each of the above regions 1)
to 6) were represented by “B7~H3 Ing”, “B7-H3 IgCl”, “B7—H3
IgV2”, “B7—H3 IgC2”, “B7—H3 IgCl—V2—C2”, and “B7—H3 IgVZ—CZ”,
respectively.
The nucleotide sequences of the ORF regions of the B7-H3
Ing, B7—H3 IgCl, B7—H3 IgV2, B7-H3 IgC2, B7—H3 IgCl—v2—C2,
and B7-H3 IgV2—C2 genes, each of which was cloned in this vector,
are represented by SEQ ID NOS: 20, 22, 24, 26, 28, and 30,
respectively, in the Sequence Listing. Further, the amino
acid sequences of the B7—H3 Ing, B7—H3 IgCl, B7—H3 IgV2, B7—H3
S l_O€SWGA/PN810023/Eng trans of PCT Spec/26.9.13
4426167—1-WARENDS
IgC2, B7—H3 IgCl—V2—C2, and B7—H3 IgV2—C2 are represented by
SEQ ID NOS: 21, 23, 25, 27, 29, and 31, respectively, in the
Sequence Listing. Further, the sequences represented by SEQ
IDNOS:ZOeNKiZIareshowninFig.15,thesequencesrepresented
by SEQ ID NOS: 22 and 23 are shown in Fig. 16, the sequences
represented by SEQ ID NOS: 24 and 25 are shown in Fig. 17, the
sequences represented by SEQ ID NOS: 26 and 27 are shown in
Fig. 18, the sequences represented by SEQ ID NOS: 28 and 29
are shown in Fig. 19, and the ces represented by SEQ ID
NOS: 30 and 31 are shown in Fig. 20.
l)—3 Production of expression vectors for B7 family genes
pCMV6—XL—4—B7RP—1, pCMV6—XL-4—B7—H1, and
pCMV6—XL—4—B7—DC (which are gene expression vectors obtained
by integrating each of B7RP—l, B7—Hl, and B7—DC (which are B7
family genes) in an expression vector pCMV6—XL—4) were all
purchased from OriGene, Inc.
s expressing each of CD80, CD86, and B7—H4, which
are B7 family genes, were produced as follows.
pENTR/221—CD80, pENTR/221—CD86, and pENTR/221—B7—H4,
which are clones ed by integrating each of CD80, CD86,
and B7—H4 in an entry vector pENTR/221, were purchased from
ogen Corporation
A Gateway LR reaction between each of the entry clones
s 107 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
whose sequence was confirmed and pcDNA3.l—DEST (Invitrogen
Corporation) was performed. For the clones obtained after
transformation of E. coli DHlOB, the size of the insert was
confirmed by colony PCR. For each of the clones in which the
size of the insert was confirmed, the DNA sequence at the 3’
end and the 5’ end of the insert was analyzed to confirm that
the insert of interest was correctly ed.
ThenucleotidesequencesoftflmaORFregions(fiftheB7RP—1,
B7—H1, B7—DC, CD80, CD86, and B7-H4 genes, each of which was
cloned in this vector, are represented.by SEQ ID NOS: 32, 34,
36, 38, 40, and 42, respectively, in the Sequence Listing.
Further, the amino acid ces of the B7RP-l, B7-H1, B7—DC,
CD80, CD86, and B7—H4 are represented by SEQ ID NOS: 33, 35,
37, 39, 41, and 43, respectively, in the Sequence Listing.
Example 2. Production ofznonoclonal antibody and.screening of
antibody
2)—1 Immunization
BALB/cAnNCrlCrlj mice (Charles River Laboratories Japan,
Inc.), chRII KO mice (Taconic, Inc., IBL Co., Ltd.), or GANP
mice (Transgenic, Inc.) at 4 to 6 weeks of age were used. On
days 0, 7, 15, and 24, LNCaP cells, MCF7 cells (ATCC) or AsPCl
cells (ATCC) detached with e (Invitrogen Corporation)
were subcutaneously administered to the dorsal region of each
mouse at a dose of 5 x 106 cells/mouse. On day 31, the same
FP1214S 108 WGA/PN810023/Eng trans of PCT 6.9.13
4426167—1-WARENDS
cells were intravenously administered to each mouse at a dose
of 5 x 106 cells. On day 34, the spleen was excised from each
mouse and used for the tion of hybridomas.
2)—2 Production of omas
Spleen.cells andxnouse myeloma P3X63Ag8U.l cells (ATCC)
were subjected.to cell fusion using PEG 4000 (manufactured by
IBL Co., Ltd.), thereby producing hybridomas.
As a result, 9639 clones from the mice immunized with
LNCaP cells, 4043 clones from the mice immunized with MCF7 cells,
and 3617 clones from the mice immunized with AsPCl cells were
established as hybridomas. By using the obtained culture
supernatant of each hybridoma, an antibody—producing
hybridoma was screened by a CDC assay.
2)—3 Screening of antibody by CDC assay
On day 0, LNCaP cells or MCF7 cells were diluted to 5000
cells per 80 HL and the resulting solution was added to a 96—well
plate at 80 ul/well. Then, the cells were ed overnight.
The hybridoma culture supernatant was added at 20 ul/well to
the plate in which the cells were seeded, and the plate was
left to stand at 4°C for 1 hour. To a diluted and lyophilized
rabbit complement (Cedarlane Laboratories), 1 mL of sterile
water was added to each vial on ice, and the vial was left to
s 109WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
stand for llninute, followed byInixing, and then the resulting
mixture was mixed with 19 mL of O. 1% BSA/RPMI 1640 medium (BSA,
Sigma Co., Ltd.). A reaction was allowed to proceed at 37°C
for 1 hour.
The plate was left at room ature for 30 minutes
toreturnix>roomtemperature. 120palofCellTiter—Gloreagent
(Promega Corporation) was added to each well, and a reaction
wasallowedtx>proceedefi1romntemperatureforll)minutes. The
amount of luminescence waSJneasured,usingaaplate reader (ARVO
HTS, PerkinElmer, Inc.). In a well exhibiting low
luminescence, it was determined that ment—dependent
cell death was induced. A hybridoma which produced a culture
supernatanttflufi:inducedsmxjicomplement-dependent.celldeath
was selected.
As a , 24 clones from the clones derived from
immunization with LNCaP, 36 clones from the clones derived from
immunization with MCF7, and 3 clones from the clones derived
fronlimmunization.with.AsPCl were obtained as positive clones
by screening.
Example 3. fication of antigen
3)—l Identification of immunoprecipitated substance
3)—l—1 Immunoprecipitation
MCF7 cells were cultured at 5 to 10 x 108 cells. These
FP1214s 110 WGA/PN810023/Eng trans of PCT 6.9.13
4426167-1—WARENDS
cells were detached with a‘cell scraper e detached cells
were collected and cryopreserved at —80°C. To the
cryopreserved cells, 10 ml of a lysis buffer which contained
1% NP—40 (Sigma—Aldrich Co., Ltd.) and a protease inhibitor
(F. Hoffmann—La Roche, Ltd.) and was cooled to 4°C was added
and the cell pellet was lysed on ice with a pipette in such
anmnnerthattheformationofkmbbleswasavoided. Afterbeing
completely lysed, the pellet was left on ice for 30 s.
The solubilized sample was centrifuged at 4°C for 20 minutes
at 10000 to 15000 rpm, and the resulting supernatant was
transferred to a 15 ml Falcon tube.
500 rm. of protein G—Sepharose 4FF beads (Amersham
Pharmacia Biotech Co., Ltd.) were washed three times, and
ted to buffer exchange with a lysis buffer. 500 pl of
theproteinG-Sepharose4FFbeadswereaddefltothesolubilized
sample supernatant on ice, and the resulting mixture was
subjected to rotary stirring overnight at 4°C.
The sample was passed h Poly—Prep chromatography
columns (Bio—Rad Laboratories, Inc.), and a passed-through
fraction was used as an immunoprecipitation sample.
3 pg of an dy solution to be used for
immunoprecipitation.was added to f protein.G—Sepharose
4FFbeadssubjectedtx>bufferexchangewithphosphatebuffered
saline (PBS) (a 1.5 ml tube), and the resulting mixture was
subjected.to rotary stirring at 4°C for 1 to 16 hours, whereby
FP1214S 111 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
the antibody was bound to the beads. To the
immunoprecipitation , the beads to which the antibody
was bound were added, and the resulting mixture was subjected
to rotary stirring at 4°C for 3 hours.
The colunwlwas transferred toeulempty lSHfl.Falcon.tube,
and 6.5 ml of a lysis buffer was added thereto. This procedure
was repeated 4 times.
The outlet of the column was capped, and pipetting was
performed with 500 pl of a lysis buffer, and the beads were
tedirlal.51mLtube. Thisprocedurewasrepeatedtwice.
After centrifugation at 4°C for 1 minute at 5000 rpm,
the supernatant was carefully removed. Then, 90 pl of an
elution buffer (10 mM glycine—HCl, pH 2.0) was added thereto,
followed by ing and centrifugation. The column of 1.5
ml spin column was detached, and 10 pl of’l M Tris—HCl (pH 8.5)
was added thereto, and the column was returned.to the original
place. An elution fraction was transferred thereto, and
centrifugationwasperformedatlOOOOrpmforILminute,whereby
100 pl of a sample was ed.
The obtained sample was subjected to MS analysis by a
liquid phase digestion technique as shown in the following
3)—l—2.
3)—l—2Identificationwafantigenku/massspectrometryanalysis
According to common ures, the fraction obtained
FP1214s 112 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
by the precipitation method was subjected to a digestion
reaction at 37°C for 16 hours by adding trypsin (modified
trypsin, Promega Corporation) through a liquid phase digestion
que. The resulting digested peptides were subjected to
a liquid chromatography (LC) /tandem mass spectrometer (MS/MS)
(Thermo Fisher Scientific K.K.) . The ed mass spectral
data were analyzed using database search software (Mascot,
Matrix Science K.K.) . As the database, International Protein
Index (IPI) was used. As a result, 34 types of ns were
identified.
From the characteristics of the identified antigens,
bibliographic information retrieval was performed based on
which B7—H3 is a cell membrane protein, and by focusing on B7—H3
(CD276) antigen (B7—H3 t 1) the experiments bed
in the following 3)—2 and 3)-3 were performed.
3) —2 Preparation of antigen gene—expressing cells
NIH—3T3 cells (ATCC) were seeded at 5 X 104 cells/cm2 in
a collagen type I~coated flask (manufactured by IWAKI Co.,
Ltd.) and cultured overnight in DMEM medium (Invitrogen
Corporation) containing 10% fetal bovine serum (FBS) under the
conditions of 37°C and 5% C02.
On the next day, the NIH—3T3 cells were transfected with
each of the pcDNA3.l-B7—H3 produced in Example l)—l—l,
DESTéO—B7—H3 variant 2 produced in l)—l—2, and
pcDNA—DEST40whichis2H1emptyvectorusingldpofectamine2000
(Invitrogen Corporation), and further cultured overnight
under the conditions of 37°C and 5% C02.
On the next day, the transfected NIH-3T3 cells were
treatedxwith trypsin, and.washed.with_DMEM containing 10% FBS,
and thereafter suspended in PBS containing 5% FBS. The thus
obtained cell suspension. was used jJi a flow cytometric
analysis.
3)—3 Flow tric analysis
The binding specificity, for B7—H3, of the antibody
ed by the hybridoma which immunoprecipitated the B7—H3
variant 1 identified by MS was confirmed by a flow cytometric
method. The cell suspension prepared in Example 3)—2 was
centrifuged, and the supernatant was removed. Then, the
hybridoma culture supernatant was added to the NIH-3T3 cells
transfectedwitheachvectortosuspendthecells,andthecells
were left to stand at 4°C for 1 hour.
After the cells were washed twice with PBS containing
% FBS, fluorescein—conjugated goat IgG on.to mouse IgG
(whole le) (manufactured by ICN Pharmaceuticals, Inc.,
#55493) diluted to lOOO—fold with PBS containing 5% PBS was
added o to suspend the cells, and the cells were left
to stand at 4°C for 1 hour.
After the cells were washed twice with PBS containing
FP1214S 114 810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
% FBS, the cells were resuspended in PBS containing 5% FBS
supplemented with 2 ug/ml 7—aminoactinomycin D (manufactured
by Invitrogen Corporation (Molecular )), and the
icnlwas performedlisingéaflOW'cytometer (FCSOO, Beckman
Coulter,Inc.). ThedatawasanalyzedusingFlowjo(TreeStar,
Inc.).
7~Aminoactinomycin tive dead cells were excluded
using a gate. Then, the FITC fluorescence intensity
histograms of viable cells were created.
A hybridoma which ed a sample that gave a higher
fluorescence intensity in the fluorescence intensity
histograms of the NIH—3T3 cells expressing the B7~H3 variant
1 and the NIH—3T3 cells expressing the B7—H3 variant 2 than
in the fluorescence intensity histogram of the NIH—3T3 cells
transfected with the empty vector serving as the control was
selected as an anti—B7—H3 antibodyeproducing hybridoma.
As a result, it was found that the antibodies derived
fronlthe anti—B7-H3 antibody-producing omas of 5 clones
(L7,L8,Lll,M30,andM3l)havecross—reactivitywiththeB7—H3
variant 1 and the B7—H3 variant 2.
3)—4 Confirmation of binding property of onal antibody
to cancer cell line
It was examined as to whether the monoclonal antibodies
FP1214S 115 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
confirmed to bind to the B7—H3 t 1 and the B7—H3 variant
2 in Example 3)—3 bind to cancer cells which overexpress the
B7—H3 variant 1 and the B7—H3 variant 2 by a flow cytometric
method in the same manner as in Example 3)—3.
In place of the transfected NIH—3T3 cells, a human breast
cancer cell line (MDA—MB~231) (ATCC) and a human lung cancer
cell line (NCI—H322) (ATCC) were used. As a result, it was
med that the establishedlnonoclonal antibodies all bind
to these cancer cell lines.
3)—5 Isotype determination of monoclonal antibody
The isotypes of the monoclonal antibodies were
determined using a Mouse monoclonal isotyping kit
(manufactured by Serotec Co. , Ltd.) . As a result, the isotypes
of the antibodies derived from the anti—B7—H3
antibody—producing omas (L7, L8, L11, M30, andlM31) were
all ined to be IgGZa.
3)—6 ation of monoclonal antibody
The monoclonal antibody was purified from the ascites
of a mouse implanted with a hybridoma or a hybridoma culture
supernatant(hereinafter,referredixJas a“startingnmterial
for antibody purification”).
The mouse ascites was prepared as follows. First,
S ]_1€SWGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
BALB/cAJcl—nu/nu (CLEA Japan, Inc.) mice at 7 to 8 weeks of
age were treated with pristane actured by Sigma Co.,
Ltd.), and after about 3 weeks, a hybridoma washed with
physiologicalsalinewasimplantedwithintheabdominalcavity
at l x 107 cells per mouse. After 1 to 2 weeks, the ascites
accumulated in the abdominal cavity was collected and
sterilizedthrougha.0.22tm1filter,and1fluaresultingnmterial
was used as a starting material for antibody purification.
The hybridoma culture supernatant was prepared using
CELLine(manufacturedknlBDBiosciences,Inc.). Theculturing
was med according to the manufacturer’s protocol except
that ell-HY Growth Medium B (manufactured by StemCell
Technologies, Inc., #03805) was used as the medium. The
ted culture supernatant was filtered through a 0.45 pm
filter, and the resulting material was used as a starting
material for antibody purification.
The antibody was ed by an affinity column obtained
by immobilizing Recombinant Protein_A rPASO (manufactured by
RepliGen Corporation) on Formyl—Cellulofine (manufactured by
aku ation) (hereinafter abbreviated as
“Formyl-Cellulofine Protein A”) or Hitrap MabSelect SuRe
(manufacturedknzGE Healthcare Bio—Sciences Corporation). In
the case of the Formyl—Cellulofine Protein A, the starting
FP1214S 117 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
material for antibody purification was diluted.to 3—fold.with
a binding buffer (3 M NaCl, 1.5 M glycine, pH 8.9), and the
resulting solution.was addedTXJa column, then, the colunm was
washed with the binding buffer, ed by elution with 0.1
M citric acid (pH 4.0). On the other hand, in the case of the
Hitrap MabSelect SuRe (GE Healthcare Corporation), the
starting material for antibody purification was added to a
column, and the column was washed with PBS, ed by elution
with 2 M Arginine—HCl (pH 4.0).
After the eluted antibody solution was neutralized, the
buffer was ged with PBS.
centrationoftheantibodywasobtainedbyeluting
the antibody bound to POROS G 20 pm Column PEEK, 4.6 mm.x 100
mm, 1.7 ml ed Biosystems, Inc.) and measuring the
absorbance (O.D. 280 nm) of the eluate. Specifically, an
antibody sample diluted with PBS was added to POROS G 20 pm
equilibrated with an equilibrating buffer (30.6 mM sodium
dihydrogen phosphate/12 aq., 19.5nw4monopotassiunlphosphate,
0.15 M NaCl, pH 7.0). Then, the column was washed with the
equilibrating buffer, and.the antibody’bound.t0'the column.was
then eluted with an eluent (0.1% (v/v) HCl, 0.15 M NaCl). The
peak area of the absorbance (O.D. 280 nm) of the eluate was
measured, and the concentration was calculated according to
the following equation:
FP1214S 118 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
tration of antibody sample (mg/ml) = (Peak area
of antibody sample) / (Peak area of reference standard (human
IgG1))><Concentrationcflfreferencestandard(Hg/ml)><Dilution
factor of sample.
Further,11MBconcentration(ifendotoxincontainedijlthe
obtained antibody was measured using Endospecy ES—50M Set
(Seikagaku Corporation, #020150) and an endotoxin reference
standard CSE—L Set (Seikagaku Corporation, 5) and was
confirmed to be 1 EU/mg or less. The resulting antibody was
used in the subsequent experiment.
Example 4. Properties of anti—B7-H3 antibody
4)—l ADCP activity
1.5 mL of ycollate was administrated in the
nal cavity of a Balb/c—nu/nu mouse (female, at 6 to 10
weeks<1fage)(CharlesRiverLaboratoriesJapan,Inc.). 5Days
thereafter, macrophages were collected from the abdominal
cavity. The macrophages were added.to a 24—well plate at 500
uL/well (1 x 105 cells/well) and cultured overnight at 37°C.
The thus ed macrophages were used as effector cells.
The labeling of NCI—H322 cells to be used as target cells
was performed using PKH26 dye labeling kit (Sigma Co., Ltd.)
The target cells were detached with TrprE (Invitrogen
Corporation) and washed twice with PBS. The cells were
FP1214s llgWGA/PNBlOOZB/Eng trans of PCT spec/26.9.13
4426167WARENDS
suspended in Diluent C at l x 107 cells/ml. PKH26 dye stock
(1 mM) was d to 8 MM with Diluent C, and immediately
thereafter, the d dye solution was added thereto in an
amount equal to that of the cell suspension. The resulting
mixture was left at room temperature for 5 minutes. Then, 1
ml<xfserun1was added thereto, and further, alnediunlwith serum
was added thereto, and washing was performed twice. The thus
prepared cells were used as the target cells.
The antibody obtained in Example 3)—6 was diluted to 20
ug/nfl,witl1a e solution. Subsequently, the target cells
obtained in Example 4)—l—l were dispensed at 2 x 106 cells/100
ul/tube and mixed. The resulting mixture was left to stand
on ice for 30 minutes. The supernatant was removed, and the
cells were washed twice with a culture solution and ded
in 500 pl of a e solution. The supernatant was removed
frmntheeffectorcells,andthecmllshavingbeentreatedwith
the antibody and suspended in the culture solution were added
thereto and mixed therewith. Then, the cells were cultured
for 3 hours in a C02 tor. Thereafter, the cells were
detached with n—EDTA and collected. To the collected
cells, an FITC—labeled anti—mouse CDllb antibody (Becton,
Dickinson and Company, Ltd.) was added, and the resulting
mixture was left to stand on ice for 30 Hdnutes. The
supernatant was removed, and the cells were washed twice with
FP1214S ]_2()WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
a culture solution. The collected cells were suspended in 300
pl of a culture solution and analyzed by FACS Calibur (Becton,
Dickinson and Company, Ltd.) . In the CDllb—positive
macrophages, a positive fraction was evaluated as
ytosis-positive cells (n=3) .
As a result, as shown in Fig. 1, L7, L8, L11, M30, and
M31 induced the phagocytosis of the 22 cells by
macrophages to give a percentage of phagocytosis of 48 . 0 i O . 9%,
52.3 i 1.1%, 57.1 i 2.5%, 61.9 i 2.1%, and 57.7 i 3.0%,
respectively. Accordingly, it was shown that L7, L8, L11, M30,
and M31 dies have ADCP activity against NCI-H322 cells.
In the same manner, commercially ble anti—B7—H3
antibodies were obtained and the ADCP activity thereof was
measured. A rat anti—human B7-H3 antibody MIH35 (eBioscience
Company), a mouse anti—human B7—H3 antibody 185504 (R&D s,
Inc.) , MIH42 (Serotec Co. , Ltd.) , and DCN70 (Biolegend y)
were obtained. It was confirmed that these antibodies bind
to B7—H3 in the same manner as in Example 3) —3. By using these
antibodies, the ADCP activity was measured by the above method.
As a result, as shown in Fig. 2, when being added at l
gig/ml, MIH35, MIH42, and DCN70 induced the phagocytosis of the
NCI—I—I322 cells by macrophages to give a percentage of
phagocytosis of 4.2%, 8.2%, and 10.8%, respectively.
FP1214S 121 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
Accordingly, it was revealed that MIH35, MIH42, and DCN7O
exhibited almost no ADCP activity.
From these s, it was shown that the M30 clones
recognizimgB7-H3obtainedtmrscreeninghaveaamarkedlyhigher
ADCP ty than the commercially available B7—H3
antibodies.
4)—2 ADCC activity
4)—2—1 Preparation of effector cells
The spleen was aseptically excised from a nude mouse
CAnN.Cg-Foxn1mVCr1Crlj (Charles River Laboratories Japan,
Inc.). The excised spleen was homogenized with two slide
glasses, and subjectedtxna hemolysis treatment using BD Pharm
Lyse(HanufacturedanBDBiosciences,Ltd.#555899). Thethus
obtained spleen cells were suspended in phenol red—free RPMI
1640 (manufactured by Invitrogen Corporation) ning 10%
Fetal Bovine Serum, Ultra—low IgG (manufactured by Invitrogen
Corporation) (hereinafter abbreviated as “ADCC ”), and
the cell suspension was passed through a cell strainer (pore
size: 40 um,1nanufacturedknzBD Biosciences, Ltd.). Then, the
viablecellswerecountedbyeatrypanbluedyeexclusionassay.
After the spleen cell suspension was centrifuged, the medium
was removed, and the cells were resuspended in the edium
at a viable cell density of 1.5 x 107 cells/ml and used as
effector cells.
FP1214s 122 WGA/PN8lOOZ3/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
4)—2—2 Preparation of target cells
B7—H3—expressing 293 cells (ATCC) and empty
vector—transfected 293 cells prepared in the same manner as
inExample3)-3weretreatedwithtrypsin,andthetreatedcells
of each type were washed with 10% ntaining RPMI 1640
(Invitrogen Corporation) and then resuspended in 10%
ntaining RPMI 1640. The cells (4 x 106 cells) of each
typewerendxedwithchromium—51(5550kBq)sterilizedthrough
a 0.22 pm filter, and labeling was performed for 1 hour under
theconditionsof37%3and5%C02. eledcellswerewashed
three times with 10% FBS—containing RPMI 1640 (Invitrogen
ation),andthecellswereresuspendedat22(105cells/ml
in the ADCC medium and used as target cells.
4)—2—3 51Cr release assay
The target cells at a cell density of 2 x 105 cells/ml
were dispensed at 50 ul/well in a 96—well U—shaped bottom
microplate.50pfl.ofM30cuianisotypecontrolantibody(mIgGZa)
(eBioscience Company) was added thereto, diluted with the ADCC
medium so that the final concentration of the antibody after
adding the effector cells was 2.5 pg/ml. Then, the plate was
left to stand at 4°C for 1 hour. Thereafter, 100 pl of the
effector cells at a cell density of 1.5 x 107 cells/ml were
added.thereto, and the cells were cultured.overnight under the
FP1214s l23WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
conditions of 37°C and.5%1302. On the next day, the supernatant
was collected in a LumaPlate (manufactured by PerkinElmer,
andgammaradiationemittedtherefrmnwasneasuredusing
a gamma counter. The percentage of cell lysis caused by the
ADCC activity was calculated according to the following
equation.
Percentage of cell lysis (%) = (A—B) / (C—B) x 100
A: Radiation count from the sample well
B: Average spontaneous radiation emission count (from
wells to which the antibody and the effector cells were not
added) (n=3). The same procedure as that for the sample well
wasperformedexceptthattheADCCnedimnwasaddedinanamwunt
of 50 pl at the time of adding the antibody and in an amount
of 100 pl at the time of adding the effector cells.
C: Average nlradiation emission count (fronlwells
in which the target cells were dissolved with a surfactant)
(n=3). The same procedure as that for the sample well was
performed except that 50 ul of the ADCC medium was added at
the time of adding the antibody and 100 pl of the ADCC medium
ning 2% (v/v) Triton X-100 was added at the time of adding
the effector cells.
ashownareeu1averageoftriplicateneasurements,
FP1214s 124 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
and the error bars represent standard.deviations. The P value
was calculated 'using Student’s t-test. The measurement
results are shown in Fig. 3.
As a result, M30 exhibited cell lysis activity with a
percentage of cell lysis of 31.6 i 3.3% against the
B7—H3—expressing 293 cells, and therefore, it was shown that
theM30antibodyhasADCCactivityagainsttheB7—H3—expressing
293 cells.
4)-3 CDC activity
An experiment was performed in the same manner as in
e 2) —3. As the cells for use in the tion, NCI-H322
cells were used. Each of the anti—B7-H3 dies (L7, L8,
L11, M30, and M31) obtained in Example 3)—6 and an isotype
control antibody (mIgG2a) diluted with 10% FBS—containing RPMI
1640 (containing antibiotics: penicillin and streptomycin)
was added so that the final concentration of the antibody after
adding complement was 25 ug/ml, e resulting mixture was
left to stand at 4°C for 1 hour. Thereto was added rabbit
complement (manufactured.by ane Laboratories, #CL3051)
diluted to 30% with RPMI 1640 so that the final concentration
of the complement was 5%, and the resulting mixture was
incubated for 1 hour under the conditions of 37°C and 5% C02.
Then, the mixture was left to stand at room temperature for
minutes. In order to measure the cell
viability, CellTiter—Glo Luminescent Cell Viability Assay
(manufactured by Promega Corporation) was added thereto in an
amount equal to that of the culture solution, and.the resulting
mixture was stirred at room temperature for 10 s.
Thereafter, the amount of scence was measured using a
plate reader. The cell viability was calculated according to
the following equation.
Cell viability (%) = (a—b) / (c—b) x 100
a: Amount of luminescence from the sample well
b: Average amount of luminescence of background (from
wells to which the cells and the antibody were not added)
(n=3) .
The same procedure as that for the sample well was med
except that an equal amount of 10% FBS—containing RPMI 1640
(containing antibiotics: penicillin and streptomycin) was
addedjxlplacecflfthecellsuspensionafl:thetimecflfcellseeding
and 10% FBS—containing RPMI 1640 (containing otics:
llin and streptomycin) was added in an amount equal to
that of the antibody dilution solution at the time of adding
the antibody.
c: Average amount of scence from wells to which
the antibody was not added (n=3). The same procedure as that
for the sample well was performed except that 10%
FBS—containing RPMI 1640 (containing antibiotics: penicillin
FP1214s 126WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
and streptomycin) was added in an amount equal to that of the
antibody dilution solution.at the time of adding the antibody.
The measurement results are shown in Fig. 4. The data
shown.are an average of triplicate measurements, and the error
bars represent standard ions. As a result, the control
antibody, L7, L8, L11, M30, and M31 d a decrease in cell
ity of the NCI-H322 cells to 101.5 i 3.3%, 6.3 i 4.2%,
13.6j:9.1%,7.2:t1.4%,7.5:t1.8%,and12.8:t2.0%,respectively,
in the presence of complement. Therefore, it was shown that
the L7, L8, L11, M30, and M31 dies have CDC activity
against the NCI—H322 cells.
4)—4 Determination of binding domain
It was examined as to which domain of B7—H3 M30 binds
by a flow cytometric method in the same manner as in Example
3)-3. The NIH—3T3 cells ected. with each of the
expression vectors for B7—H3 partial proteins prepared in
Example 1)-1—3 were used.
As a result, as shown in Fig. 5, it was confirmed that
M30bindstoB7—H3IgC1,B7—H3IgC2,B7-H3IgC1—V2—C2,andB7—H3
IgVZ—CZ. M30 did not bind to B7—H3 IgV1 and B7—H3 IgV2.
From these results, it was shown that M30 binds to the
C1 domain (the amino acid sequence represented by amino acid
numbers 140 to 244 in SEQ IDIMD:6) and the C2<domain (the amino
FP1214S 127 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
acid sequence represented by amino acid numbers 358 to 456 in
SEQ ID NO: 6) of B7—H3. In the same manner, it was also shown
that L8, L11, and M31 bind to the C1 domain and the C2 domain,
and L7 binds to the V1 domain (the amino acid sequence
ented by amino acid numbers 27 to 139 in SEQ ID NO: 6)
and the V2 domain (the amino acid sequence represented by amino
acid s 245 to 357 in SEQ ID NO: 6).
Accordingly,:fl:was showniflufl:M30 recognizesemiepitope
in the IgC1 domain and/or the IgC2 domain, each of which is
a domain in the B7—H3 ellular domain, and binds to the
IgCl domain or the IgC2 domain or both.
4)—5 Antigen specificity
The antigen specificity of M30 was examined by a flow
tric method in the same manner as in Example 3)—3.
293T cells transfected with each of the expression
vectors for the CD80, CD86, B7—RP—1, B7~H1, B7—DC, and B7—H4
proteins, which are B7 family proteins, prepared in Example
1)—1—4 were used.
As a result, it was shown that M30 does not bind to CD80,
CD86, B7—RP-1, B7—H1, B7—DC, and B7—H4, which are B7 family
molecules.
Example 5. In Vivo antitumor effect
F91214s 1428 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
)—l In vivo antitumor effect of anti—B7—H3 antibody
22 cells were detached from a culture flask by a
trypsin treatment, and then suspended in 10% PBS—containing
RPMI 1640 (Invitrogen Corporation), followed by
centrifugation, and the supernatant was removed. The cells
were washed twice with the same medium, and then suspended in
physiological saline (manufactured by Otsuka Pharmaceutical
Co., Ltd.). Then, the cells were implanted subcutaneously in
theaxillaryregion<xfeachofeagroup<xfBALB/cAJcl—nu/nu(CLEA
Japan, Inc.) mice at 6 weeks of age at a dose of l x 107
cells/mouse. The day of implantation was taken as day 0, and
on days 10, 17, 24, 31, and 38, each of the L7, L8, L11, M30,
andM3lantibodieswasintraperitoneallyadministeredataadose
of 500 ug/mouse (about 25 mg/kg). To the control, PBS was
intraperitoneally stered in a volume (500 ul) equal to
that of the antibody. The tumor volume was measured on days
, 17, 24, 31, 38, and 45, and the antitumor effect of the
administration of the antibody was examined.
As a result, in the M30 and M31 administration groups,
the tumor growth was significantly suppressecias comparedivith
the PBS administration group. (The P values for M30 and M31
comparedwith1flmaPBSadministrationgroupjxltermscfifthetumor
volume on day 45 were P<0.05 and , respectively. The
P values were calculated using Student’s t-test.). Further,
thetumorgrowthinhibitionratio (averagetumorvolume
FP1214S 1229 WGA/PNBlOOZB/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
in antibody administration group) / (average tumor volume in
PBS administration group) x 100) on day 45 in the case of L7,
L8,L11,M30,andM3lwas—16.l%,0.2%,25.5%,47.2%,and58.2%,
respectively. Accordingly, the M30 and M31 antibodies were
observed to have a very strong antitumor effect in vivo (Fig.
From the above results, it was revealed that the M30 and
M31 antibodies are antibodies which recognize a B7-H3 antigen
and exhibit an antitumor effect.
)—2 In vivo antitumor effect under ions of depletion
of macrophages
In order to deplete macrophages in Vivo,
clodronate—encapsulatedliposomesweregmoduced. Ifl:hasbeen
reported that by stering nate—encapsulated
liposomes in vivo, macrophages in vivo are depleted (Journal
of immunological methods 1994, vol. 174, pp. 83—93).
According to the method in this report,
clodronate—encapsulated liposomes were ed and used in
the following experiment.
NCI—H322 cells were detached from a culture flask by a
trypsin treatment, and then suspended in 10% FBS—containing
RPMI 1640 (Invitrogen Corporation), followed by
FP1214s /PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
centrifugation and the supernatant was removed. The cells
were washed twice with the same , and then suspended in
physiological saline (PBS, manufactured by Otsuka
Pharmaceutical Co., Ltd.). Then, the cells were implanted
subcutaneouslyjjltheaxillaryregion<xfeachBALB/cAJcl—nu/nu
(CLEA Japan, Inc.) mouse at 6 weeks of age at a dose of 1 x
107 cells/mouse. The day of implantation was taken as day —14,
and grouping was performed on day 0.
In a group in which the macrophages in vivo in the mice
were to be ed, the clodronate—encapsulated liposomes
were intravenously injected at a dose of 0.2 mL/mouse on days
0, 4,7, 11,14, 18,21, 25,28, and32. Further,j11thenegative
control group, PBS was intravenously injected at a dose of 0.2
mL/mouse on the same days (on days 0, 4, 7, 11, 14, 18, 21,
, 28, and 32).
uently, the M30 antibody was intraperitoneally
administered to both groups at a dose of 500 ug/mouse (about
mg/kg) on days 1, 8, 15, 22, and 29. Further, as the negative
control, PBS was intraperitoneally administered to both groups
in a volume (500 pl) equal to that of the M30 antibody on the
same days (on days 1, 8, 15, 22, and 29).
The tumor volume was measured on days 0, 8, 15, 22, 29,
and 36, and the mor effect of the administration of the
antibody was examined (n=8).
The results are shown in Tables 1 and 2, and Fig. 7.
FP1214s 1.3]_WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
[Table l]
endavo Ways
Tumor volume Average Standard Average Standard Average Standard
(mm3) deviation (mm3) deviation (mm3) deviation
.P.BS +.PBS 104.375 6491581086 149 10.60828517 227.5 18.20027472
administration group
C.|0.d hp + PBS 114.625 4.862162585 159.375 7676349346 318.625 2637567704
stration group
.P.BS +.M30 103.5 7.221001118 123.625 14.89958952 145 19.58497967
administration group
(3.10.“pr M30 104.625 5.47049717 143.375 1046753058 247.75 47414
administration group
On day 22 On day 29 On day 36
Tumor volume e Standard Average Standard Average Standard
(mm3) deviation (mm3) ion (mm3) deviation
.PEBS +.PBS 394.75 72433 601.25 43.17065389 827 5082638516
administration group
C.'°.d 1.5+ PBS 443.625 23.52653327 619.75 409550058 1002.75 78.18493415
administration group
.P.BS +.M3O 186.25 25.920035 301.75 47.13610612 415.25 79.84175197
administration group
C.lo.dhp .+ M30 384.25 40.10644319 641.375 80.12176838 837.25 121349223
stration group
In the group of PBS intravenous administration + M30
antibody intraperitoneal administration (PBS + M30
administration group), the tumor growth was significantly
ssed as compared with the group of PBS intravenous
administrationi—PBSintraperitonealadministration(PBS-iPBS
administration group) serving as the negative l. To be
more specific, the P value for the PBS + M30 administration
grout>compareciwith_the PBSi—PBS administration group in terms
of the tumor volume on day 36 was P<0.05 (The P value was
FP1214S 132 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
calculated using Student’s t—test.). Further, the tumor
growth tion ratio (= 100 — (average tumor volume in PBS
+ M30 administration group) / (average tumor volume in PBS +
PBS administration group) x 100) on day 36 was 49.8% (Table
On the other hand, in the group of
clodronate-encapsulated liposome intravenous administration
+ PBS intraperitoneal administration (Clod lip + PBS
administration group) and the group of
clodronate-encapsulated liposome intravenous administration
+ M30 antibody intraperitoneal administration (Clod lip + M30
administration group), the suppression.of tumor growtrlwas not
observed. To be more specific, the P values for the Clod lip
-FPBSadministrationgroupandtheCflodlip-PM30administration
group comparedxwith the PBS—FPBS administratituigroup in terms
ofthetumorvolumeoncku736wereP=O.52andP=l,respectively.
(The P values were calculated using Student’s t-test).
Further, the tumor growth inhibition ratios (= 100 — ge
tumor volume in Clod lip + PBS administration group or Clod
lir>+ M30 administration.group) / (average tumor volume in PBS
+ PBS administration group) X 100) on day 36 were —2l.2% and
—l.4%, respectively (Table 2).
FP1214s /PN810023/Eng trans of PCT 6.9.13
4426167—1—WARENDS
[Table 2]
Tumor growth
inhmnmnrafio % Onday8 Onday15 Onday22 Onday29 Onday36
C'Od “p + PBS
-39 4' -12 6 -21 2
administration group ' -3 5' '
PBS +.M30 17.5 36.4 52.8 49.7 49.8
stration group
cmwm+Mm
32 26 44
administration oroup '
' ‘
From the above results, it was shown that the antitumor
effect of the M30 antibody was ssed by administering
nate-encapsulated liposomes, and therefore, it was
ed that the antitumor effect of the M30 antibody is mainly
an effect mediated by macrophages.
Example 6. Cloning of mouse antibody M30 cDNA and determination
of sequence
6) —1 Determination of N—terminal amino acid sequences of heavy
and light chains of mouse antibody M30
In order to determine the N—terminal amino acid sequences
of the heavy and light chains of the mouse antibody M30, the
mouse antibody M30 purified in Example 3)—-6 was ted by
SDS—PAGE. The protein in the gel was transferred from the gel
after separation to a PVDF membrane (pore size: 0.45 um,
manufactured by Invitrogen Corporation). The membrane was
washed with a washing buffer (25 mM NaCl, 10 mM sodium borate
buffer pH 8.0), and thereafter stained by being immersed in
FP1214s 134 WGA/PN8lOO23/Eng trans of PCT spec/26.9.13
4426167WARENDS
a dye solution (50% methanol, 20% acetic acid, 0.05% Coomassie
brilliant blue) for 51ninutes, followed.by destaining with 90%
methanol. The portions of the band.correspondingtx>the heavy
chain (the band with smaller mobility) and the band
corresponding‘ to the light chain (the band, with larger
mobility) visualized on the PVDF membrane were excised.
The portion.of the band.corresponding to the light chain
was incubated at 37°C for 30 minutes in a small amount of a
0.5% polyvinylpyrrolidone/lOO mM acetic acid on,
followed by washing well with water. Subsequently, modified
N—terminal residue was d using Pfu utamate
AminopeptidaseKit(TaKaRaBio,Inc.),followedbywashingwith
water and air drying. Then, an attempt was made to identify
their respective N—terminal amino acid sequences by an
automaticEdmanHethod(seeEdmanetal.(l967)Eur.J.Biochem.
l, 80) using Procise (registered. trademark) cLC Protein
Sequencer Model 492cLC (Applied Biosystems, Inc.).
As a result, the N-terminal amino acid sequence of the
band corresponding to the heavy chain of the mouse antibody
M30 was determined to be EVQLQQSGPE (SEQ ID NO: 44 in the
Sequence g), and the N—terminal amino acid sequence of
the band ponding to the light chain f was
determined.to be IVLSQSPTILSASP (SEQ ID NO: 45 in the Sequence
Listing).
FP1214S 135 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
6)—2 Preparation of mRNA from mouse antibody M30-producing
hybridoma
In order to clone cDNAs encoding each of the heavy chain
andthelightchamiofthenwuseantibodyM30,mRNAwaspmepared
from the mouse antibody MBO—producing hybridoma using Quick
Prep mRNA Purification Kit (GE Healthcare Corporation).
6)—3 Cloning of mouse antibody M30 cDNA and determination of
sequence
With reference to the findings that the isotypes of the
heavy and light chains of the mouse antibody M30 are y2a and
K found in Example 3) ~5, and the inal amino acid sequences
of the heavy and light chains determined in Example 1—1), and
the database of the amino acid sequences of dies (see
Kabat, E. A. et al., (1991) in Sequences of Proteins of
ImmunologicalInterestVol.filandlfly U.S.Department<1fHealth
and Human Services), several oligonucleotide primers
hybridizing to each of the 5'—terminal region of an dy
gene coding region and the 3’-terminal region thereof
comprising a stop codon were synthesized, and a cDNA ng
the heavy chain and a cDNA encoding the light chain were
amplified using the mRNA prepared in Example 6—2) and TaKaRa
One Step RNA PCR Kit (AMV) (TaKaRa Bio, Inc.). As a result,
the cDNA encoding the heavy chain of the antibody and the cDNA
encoding the light chain of the antibody could be amplified
FP1214S ]_3€3WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
by the following primer sets.
Primer set for the heavy chain
Primer 16
aattcatggaatggagttggata—3’ (SEQ ID NO: 46 in the
Sequence Listing)
Primer l7
’—aagatatctcatttacccggagtccgggagaa~3’ (SEQ ID NO: 47 in the
Sequence Listing)
Primer set for the light chain
Primer l8
’—aagaattcatggattttctggtgcag—3’ (SEQ ID NO: 48 in the
Sequence Listing)
Primer l9
’—aagatatcttaacactcattcctgttgaagct—3’ (SEQ ID NO: 49 in the
Sequence Listing)
Each of the cDNA encoding the heavy chain and the cDNA
encoding the light chain amplified by PCR was cloned using
pEF6/V5—His TOPO TA sion Kit (Invitrogen Corporation),
and each of the nucleotide sequences of the heavy chain and
the light chain cloned was determined using a gene sequence
analyzer (“ABI PRISM 3700 DNA Analyzer; Applied Biosystems”
or “Applied Biosystems 3730xl.Analyzer; Applied Biosystems”).
In the sequencing reaction, GeneAmp 9700 (Applied Biosystems,
Inc.) was used.
The ined nucleotide sequence of the cDNA encoding
FP12145 137 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1~WARENDS
the heavy chain of the mouse antibody M30 is represented by
SEQ ID NO: 50 in the Sequence Listing, and the amino acid
sequence f is represented by SEQ ID NO: 51. Further,
the sequences of SEQ ID NOS: 50 and 51 are shown in Fig. 21.
The determined.nucleotide sequence of the cDNA.encoding
the light chain of the mouse antibody M30 is represented by
SEQ ID NO: 52 in the Sequence Listing, and the amino acid
sequence thereof is ented by SEQ ID NO: 53 in the Sequence
Listing. The sequences of SEQ ID NOS: 52 and 53 are shown in
Fig. 22.
r, the aminC)acid sequences of the heavy chain and
thelightchainwereanalyzaibycomparisonusingKabatMan(see
PROTEINS: Structure, on and Genetics, 25 (1996),
130—133), which is the database of the amino acid sequences
of antibodies. As a result, it was found that in the heavy
chain of the mouse antibody M30, the amino acid sequence
represented by amino acid numbers 20 to 141 in SEQ ID NO: 51
in the Sequence Listing is the variable region. It was also
found that in the light chain of the mouse dy M30, the
amino acid sequence represented by amino acid numbers 23 to
130 in SEQ ID NO: 53 in the Sequence Listing is the variable
region.
Example 7. Production.of chimeric antibodyflfl30 (cM3O antibody)
7)—l Construction of chimeric and humanized light chain
expression vector pEFGKCL
By performing PCR using a plasmid pEFG/VS—HisB
rogen Corporation) as a template and also using the
following'primers, a DNA fragment from immediately downstream
of BGHpA (Sequence Position; 2174) tc>SmaI (Sequence Position:
2958) (a DNA.fragment comprising fl origin.of replication and
SV40jpromoter‘andmariginq hereinafter edtxaas ent
A") was obtained.
Primer 2O
’—ccacgcgccctgtagcggcgcattaagc—3’ (SEQ ID NO: 54 in the
Sequence Listing)
Primer 21
'—aaacccgggagctttttgcaaaagcctagg—3’ (SEQ ID NO: 55 in the
Sequence Listing)
The obtained fragment A and a DNA fragment (SEQ ID NO:
56, hereinafter referred to as “fragment B”, the sequence of
SEQ ID NO: 56 is also shown in Fig. 23) sing a DNA sequence
encoding a human K chain secretory , a human K chain
constant region, and a human poly—A additional signal were
ligated to each other by overlap ion PCR. The thus
obtained.DNA.fragment in which.the fragment A.and.the fragment
B were ligated to each other was digested.with the restriction
enzymes KpnI and SmaI, which was ligated to a plasmid
pEF6/V5—HisB (Invitrogen.Corporation) whicfllwas digestedxnith
the restriction enzymes KpnI and SmaI, whereby a chimeric and
humanizedlightchainexpressionvectorpEF6KCLhavingeasignal
sequence, a cloning site, a cchain constant region, and
a human poly-A additional signal sequence ream of the
EFl promoter was constructed.
7)—2 Construction of pEFlKCL
A.DNA nt obtaineciby cleaving the pEF6KCl.obtained
bytheabove—describednethodwiththerestrictionenzymestnI
andSmaIwasligatedtopEFl/myc—HisB(InvitrogenCorporation)
whichwasdigestedwitthnIandSmaI,wherebyagflasmidpEFlKCL
was constructed.
7)—3 Construction of chimeric and humanized heavy chain
expression vector pEFlFCCU
A DNA fragment (SEQ ID NO: 57, the sequence of SEQ ID
NO: 57 is also shown in Fig. 24) comprising a DNA sequence
ng amino acids of a signal sequence and a constant region
of human IgGl was digested with the restriction enzymes NheI
and PmeI and was ligated to the plasmid pEFlKCL which was
digested with NheI and PmeI, whereby a chimeric and humanized
heavychainexpressionvectorpEFlFCCUhavingeasignalsequence,
acloningsite,eahumanheavychainconstantregion,andeahuman
poly—A onal signal sequence downstreanl of the EFl
FP1214s 140 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
promoter was constructed.
7)—4 Construction of M30 chimera—type light chain expression
vector
By using the cDNA encoding the light chain of the mouse
antibody M30 as a template and also using KOD—Plus— O,
Co., Ltd.) and the following primer set, a region comprising
thecDNAencodingthelightchainvariableregbmiwasamplified.
A DNA fragment obtained.by cleaving the amplified product with
the restriction enzymes NdeI and BsiWI was inserted into the
universal ic and humanized antibody light chain
expression vector (pEF6KCL) at the site cleaved with the
restrictionenzymesNdeIanstiWI,wherebyanMBOchimera-type
light chain expression vector was constructed. The thus
obtained expression vector was named “pEF6KCL/M3OL”. The
nucleotide ce of the M30 a—type light chain is
represented by SEQ ID NO: 58 in the Sequence Listing, and the
amino acid sequence thereof is represented by SEQ ID NO: 59.
The sequences of SEQ ID NOS: 58 and 59 are shown in Fig. 25.
ntally, a threonine residuezat position 128 in the amino
acid sequence of the cM30 antibody light chain represented by
SEQZHDNO:59:UltheSequenceListingislocatedjjithecarboxyl
terminus of the light chain variable region and corresponds
UsanalanineresidueatpmsitionlBOjjltheaminoacidsequence
of the M30 antibody light chain represented by SEQ ID NO: 53
FP1214s l41WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
in the ce Listing, however, in the amino acid sequence
represented by SEQ ID NO: 59, the residue has already been
substituted with a threonine residue derived from a human
antibody light chain.
Primer set for the light chain
Primer 22
catatggccaaattgttctctcccagtctccaacaatcc—3’ (SEQZUDNO:
60 in the Sequence Listing)
Primer 23
'-aaacgtacgtttcagctccagcttggtcccagtaccg-3’ (SEQIUDNO: 61in
the Sequence Listing)
7)—5 Construction of M30 chimera—type heavy chain sion
vector
By using the cDNA encoding the heavy chain of the mouse
antibody' M30 as a template, a DNA fragment obtained by
ming PCR using KOD-Plus— (TOYOBO, Co., Ltd.) and the
following primer set A was ligated to a DNA fragment obtained
by performing PCR using the following primer set B through
p extension PCR using the following primer set C, whereby
the Blpl in the variable region was removed and also a region
comprising the CDNA encoding the heavy chain variable region
was amplified. A DNA fragment obtained by cleaving the
amplifiedproductwiththerestrictionenzymeBlprasinserted
intothelnflyersalchimericandhumanizedantibodyheavychain
FP1214S 142 WGA/PNBlOOZB/Eng trans of PCT spec/26.9.13
4426167WARENDS
expression vector (pEFIFCCU) at the site cleaved with the
restriction enzyme BlpI, whereby an M30 chimera—type heavy
chain expression vector was constructed. The thus obtained
expression vector was named “pEFlFCCU/M3OH”.
The nucleotide sequence of the M30 a—type heavy
chain is represented by SEQ ID NO: 62 in the ce Listing,
and the amino acid sequence thereof is represented by SEQ ID
NO: 63. Further, the sequences of SEQ ID NOS: 62 and 63 are
shown in Fig. 26.
Primer set A
Primer 24
’—aaagctgagcgaggtccagctgcagcagtctggacctgag—3’ (SEQ ID NO:
64 in the Sequence Listing)
Primer 25
’—gaggtcaggctgctgagttccatgtaggctgtgctg—3' (SEQ ID NO: 65 in
the Sequence Listing)
Primer set B
Primer 26
’—cagcacagcctacatggaactcagcagcctgacctc-3’ (SEQ ID NO: 66 in
the Sequence Listing)
Primer 27
gctgagctgactgtgagagtggtgccttggccccag-3’ (SEQ IDDKM 67
in the Sequence Listing)
Primer set C
Primer 28
FP1214s ]_423wsA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
’—aaagctgagcgaggtccagctgcagcagtctggacctgag—3’ (SEQ ID NO:
68 in the Sequence Listing)
Primer 29
’—aaagctgagctgactgtgagagtggtgccttggccccag—3’ (SEQ IDDKM 69
in the Sequence Listing)
7)—6 Preparation of chimeric antibody M30
7)-6—l Production of ic antibody M30
1.2 x 109 cells of FreeStyle 293E cells (Invitrogen
Corporation) in the logarithmic growth phase were seeded into
1.2 L of fresh FreeStyle 293 Expression Medium (Invitrogen
Corporation) and cultured for 1 hour by shaking at 90 rpm at
37°C in an 8% C02 incubator. 3.6 mg of polyethyleneimine
(Polyscience #24765) was dissolved in 20 ml of Opti—Pro SFM
medium (Invitrogen Corporation). Subsequently,
pEFlFCCU/M3OH(O.4Imfl andpEF6KCL/M3OL(O.8mg)preparedwith
PureLink HiPure Plasmid Kit (Invitrogen Corporation) were
ded in 20 ml of Opti—Pro SFM medium” Then, 20 ml of the
obtained expression vectors/Opti—Pro SFM mixed liquid was
to 20 ml of the obtained polyethyleneimine/Opti—Pro SFM
mixed.liquid, and the ingxnixture was gently stirred.and
then left for 5 minutes. Thereafter, the mixture was added
to the FreeStyle 293F cells, and shaking culture at 90 rpnlwas
performed for 7 days at 37°C in an 8% C02 incubator. The
resulting culture atant was filtered through a
FP1214s 144 810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
disposable capsule filter (Advantec #CCS—O45—E1H).
A chimeric dy M30 obtained by a combination of
pEFlFCCU/M3OH and pEF6KCL/M3OL was named “cM30” or “cM3O
antibody”.
7)—6—2 Purification of cM3O
The e supernatant obtained in Example 7)—6—l was
purified by a two-step process including rProtein A affinity
tography (at 4 to 6°C) (GE care Japan Corporation)
and ceramic hydroxyapatite (at room temperature). A buffer
exchange step after the purification by rProtein A affinity
chromatography and after the purification by ceramic
hydroxyapatite was performed at room temperature. First, 1100
to 1200 ml of the culture supernatant was applied.to MabSelect
SuRe(HanufacturedkanEHealthcareBio-SciencesCorporation,
two HiTrap columns (volume: 1 ml) connected. in series)
equilibrated with PBS. After all e solution was poured
into the column, the column.was washed with 15tx>30 ml of PBS.
Subsequently, elution was performed with a 2 1% arginine
hydrochloride solution (pH 4 . O) a fraction containing the
, and
antibody was collected. The fraction. was d. to a
desalting column (manufactured.by GE Healthcare Bio—Sciences
Corporation, two HiTrap desalting columns (volume: 5 ml)
ted in series), whereby the buffer was exchanged with
a buffer containing 5 mM sodium phosphate, 50 mM MES, and 20
mM NaCl at pH 6.5.
Further, the antibody solution ted to buffer
ge was appliedixna.ceramic hydroxyapatite column (Japan
Bio—Rad Laboratories, Inc., Bio—Scale CHTZ-l hydroxyapatite
column (volume: 2 ml)) equilibrated with a buffer containing
mM NaPi, 50 mM MES, and 20 mM NaCl at pH 6.5. Then, linear
concentration. gradient elution with sodiunl chloride was
performed,andeafractioncontainingtheantibodywascollected.
The fraction was applied to a desalting column (manufactured
by GE Healthcare Bio—Sciences Corporation, two HiTrap
Desalting columns e: 5nflj connectedjjlseries), whereby
the liquid was exchanged with CBS (containing 10 mM e
buffer and 140 mM sodium chloride, pH 6.0).
Finally, the resulting solution was concentrated using
CentrifugalUFEfilterDeviceVIVASPINZO(fractionalnwlecular
weight: 30 K, Sartorius Co., Ltd., at 4°C), and the
concentration of IgG was adjusted to 1.0 mg/ml or more, and
the thus obtained solution was used as a purified sample.
Example 8. Activity of cM30 antibody
8)—l Binding activity of cM30 antibody to B7—H3
The ty between the M30 antibody or the cM3O
antibodyandtheB7—H3antigenwasneasuredknzasurfaceplasmon
resonance (SPR) device (GE Healthcare Corporation).
According to common procedures, an anti—mouse IgG or an
FP1214s l46WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
anti—human IgG antibody was immobilized.on a sensor chip, and
then, the M30 antibody sample or the cM30 antibody was bound
thereto. Thereafter, an ellular domain polypeptide of
a recombinant B7—H3 variant 2 antigen (manufactured by R&D
Systems,Inc.,#2318—B3-050/CF)wasaddedtheretoatchfferent
concentrations, and the amount of the antigen bound to the
antibody in a running buffer (phosphate buffer, 0.05% SP20)
wasrneasured.over time. The1neasured.amount was ed.with
dedicated software (BIAevaluation.Version 4.1, GE Healthcare
Corporation) and a dissociation constant was calculated.
As a result, the M30 antibody and.the cM3O antibody were
found to have bound to the recombinant B7-H3 antigen with a
dissociation nt of 5.89 nM and 3.43 nM, respectively.
From these results, it was med that the M30 antibody'and
the CM3O dy bind to the B7—H3 antigen and their binding
affinities were substantially equal.
8)—2 ADCP activity of cM3O antibody
Peripheral blood.mononuclear cells (PBMCs) of a.healthy
subject were isolated according to common procedures and
suspended in 10% FBS-containing RPMI 1640 (Invitrogen
Corporation) and then seeded in a flask. The cells were
edovernightjilacogincubator. Theculturesupernatant
was removed, and to the cells attached to the flask, 10%
FBS—containing RPMI 1640 supplemented with M—CSF and GM—CSF
(PeproTech, Inc.) was added, and the cells
werecnflturedfOrZZweeks. ThecellsweredetachedwithTrprE
and collected. Then, the cells were added to a 24—well plate
at 500 ul/well (l x 105 cells/well) and cultured ght at
37°C. The thus prepared cells were used as effector cells.
The labeling of NCI-H322 cells to be used as target cells
was performed.using PKH26 dye labeling kit (Sigma Co., Ltd.).
The target cells were detached with TrprE and washed twice
with PBS. The cells were suspended in Diluent C at l x 107
cells/ml. PKH26 dye stock (1 mM) was diluted to 8 uM with
Diluent C, and immediately thereafter, the diluted dye solution
was added thereto in an amount equal to that of the cell
suspension. The resulting mixture was left at room
temperature for 5 minutes. Then, 1 ml of serum was added
thereto, and further, a medium with serum was added thereto,
and washing was performed twice.
EadioftheM30antibodyandtheCMBOantibodywasdiluted
tc>20 ug/ml witheaculture solution. Subsequently, the target
cells were dispensed at 2 x 106 cells/100 ul/tube and mixed.
The resulting e was left to stand on ice for 30 minutes.
The supernatant was d, and the cells were washed twice
with a culture solution and suspended in 500 pl of a culture
solution. The supernatant was removed fronlthe effector cells,
and the cells having been treated with the antibody and
ded in the culture solution were added thereto andlnixed
FP1214S 1g48 810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
therewith. Then, the cells were cultured for 3 hours in a C02
incubator. fter, the cells were ed with
Trypsin—EDTA. and ted. To the collected cells, an
FITC—labeled.anti—mouse CD11b antibody (Becton, Dickinson and
Company, Ltd.) was added, and the resulting mixture was left
to stand on ice for 30 minutes. The supernatant was removed,
and the cells were washed twice with a culture solution. The
collected cells were suspended in 300 ul of a culture medium
and analyzed by FACS Calibur (Becton, Dickinson and Company,
Ltd.). In the CDllb—positive macrophages, a PKH26—positive
fraction was evaluated as phagocytosis—positive cells.
As a result, as shown in Fig. 8, when the M30 antibody
and the cM3O antibody were added at 10 ug/ml, the phagocytosis
of the NCI—H322 cells by hages was induced to 33 i 1%
and 35:t2%, respectively. Accordingly, it was shown that the
cM3O antibody has an ADCP activity against the NCI-H322 cells
unthesamenannerasijuaMBOantibody. leimilarexperimental
result was obtained. also for an ADCP activity against
MDA-MB-23l cells (ATCC).
8)—3 In vivo antitumor effect of cM30 antibody
MDA—MB—231 cells were detached from a e flask by
a trypsin treatment, and then suspended in 10% PBS-containing
RPMI 1640 . (Invitrogen Corporation), followed by
FP1214S sA/pN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
centrifugation and the supernatant was removed. The cells
were washed twice with the same medium, and then suspended in
BD Matrigel Basement ne Matrix actured by BD
Biosciences, Inc.). Then, the cells were implanted
subcutaneously in the axillary region of each mouse
(CB17/Icr—Prkdc[scid]/CrlCr1j, Charles River Laboratories
Japan, Inc.) at 6 weeks of age at a dose of 52<1IW cells/mouse.
The day of implantation was taken as day 0, and on days 14,
21, 28, 35, and 42, the M30 antibody or the cM3O antibody was
intraperitoneallyadministeredataadosecfif500ug/mouse(about
. The tumor volume was measured on days 14, 18, 21,
, 28, 32, 35, 39, 42, 46, 49, and 52, and'theiantitumor‘effect
of the administration of the antibody was examined.
Asearesult, iniflueM3O and.cM3O antibody administration
groups, the tumor growth was significantly suppressed as
compared with the untreated group in which the antibody was
not administered. The P values for the M30 antibody and the
cM3O dy compared with the untreated group in terms of
the tumor weight on day 52 were both P<0.001. The P values
were calculated using Dunnett's multiple comparison test.
Further, the tumor growth inhibition ratio (= 100 —
ge tumor volume in antibody administration group) /
(average tumor volume in untreated group) x 100) on day 52 was
71.3% in the case of the M30 dy and 71.7% in the case
of the cM3O antibody. Accordingly, the cM3O antibody was
FPl2l4s 150 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
observed to have a very strong mor effect in vivo in the
same manner as the M30 dy (Fig. 9).
Examplefk Designingoflumwnizedantibodycflfmouseanti—human
B7—H3 antibody #M3O
9)—1 Designing of humanized M30 antibody
9)-1—1 Molecular modeling of M30 variable regions
The molecular modeling of the M30 variable regions was
performed according to a method generally known as homology
ng (Methods in Enzymology, 203, 121—153, (1991)). The
primary sequences (three—dimensional structures derived from
the X—ray crystal structures are available) of the le
regionsofhumanimmunoglobulinregisteredinProteinDataBank
(Nuc. Acid Res. 35, DBOl—D303 (2007)) were compared with the
M30 variable regions determined in Example 6—3).
As a result, 3BKY was selected as a sequence having the
highest sequence homology with the M30 light chain variable
region among the antibodies which similarly have a deletion
in the framework. Further, 3DGG was selected as a sequence
having the highest sequence homology with the M30 heavy chain
variable region.
The three—dimensional structure of a framework region
was ed based on a “framework model” by combining the
coordinates of 3BKY corresponding to the M30 light chain with
the coordinates of 3DGG corresponding to the M30 heavy chain.
S 151WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
As for the M30 CDRs, as the coordinates defining the
conformations most similar to those of CDRHl (SEQ ID NO: 92),
CDRHZ (SEQ ID NO: 93), CDRH3 (SEQ ID NO: 94), CDRLl (SEQ ID
NO: 95), CDRL2 (SEQ ID NO: 96), and CDRL3 (SEQ ID NO: 97)
according to the classification of on et al. (J. Mol.
Biol., 263, 800—815, (1996)) and the H3 rules (FEBS letters
399, 1—8 (1996)), 2HOJ, lBBD, 1Q90, ZFBJ, lLNK, and lTET were
selected, respectively, and incorporated in the framework
model.
Finally, in order to obtain a possible molecular model
of the M30 variable region in terms of energy, an energy
calculation was performed for excluding disadvantageous
interatomic contact. The above procedure was carried out
using the cially ble protein tertiary structure
prediction program Prime and the coordinate search program
MacroModel dinger, LLC).
9)-1—2 Designing of amino acid sequence of humanized M30
A humanized M30 antibody was constructed according to
a method generally known as CDR grafting (Proc. Natl. Acad.
Sci. USA 86, 10029-10033 (1989)).
Anacceptorantibodywasselectedbasedontheaminoacid
gy within the framework region. The sequence of the
framework region of M30 was compared with all human framework
s 152 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
sequences in the Kabat Database (Nuc. Acid Res. 29, 205—206
(2001)) of antibody amino acid sequences. As a result, a
mAb49‘C11antibody'(GenBankileCBI: D16838.1.and]316837.1) was
selected as an acceptor based on a sequence homology of 70%
in the framework region.
The amino acid residues in the framework region of
mAb49'CL were aligned.with the amino acid residues of M30, and
the positions where different amino acids were used were
identified. The positions of these residues were analyzed
using the three-dimensional model of M30 constructed above.
Then, donor es to be grafted onto the acceptor were
selected according to the criteria ed by Queen et al.
(Proc. Natl. Acad. Sci. USA 86, 10029—10033 (1989)). By
transferring some selected donor residues to the acceptor
antibody, zed M30 antibody sequences were constructed
as described in the following Example.
9)-2 Humanization of M30 heavy chain
9)—2-1 M30—H1—type heavy chain:
A humanized M30 heavy chain designed by tuting
amino acid s 20 (glutamic acid), 24 (glutamine), 28
(proline), 3O (leucine), 31 (valine), 35 (alanine), 39
(methionine), 57 e), 59 (lysine), 67 (isoleucine), 86
(lysine), 87 (alanine), 89 (glutamine), 91 (serine), 93
(lysine), 95 (serine), 106 (threonine), 110 (serine), 136
nine), and. 137 (leucine) of the cM30 heavy chain
represented by SEQ ID NO: 63 in the Sequence Listing with
glutamine, valine, alanine, valine, lysine, serine, valine,
arginine, alanine, methionine, ne, valine, isoleucine,
alanine, glutamic acid, threonine, arginine, threonine,
leucine, and valine, respectively, was named “M30—H1—type
heavy chain”.
The amino acid sequence of the M30-Hl~type heavy chain
is represented by SEQ ID NO: 85.
9)—2—2 M30—H2—type heavy chain:
A humanized M30 heavy chain designed by substituting
amino acid numbers 20 (glutamic acid), 24 (glutamine), 28
(proline), 3O (leucine), 31 (valine), 35 (alanine), 39
onine), 57 (lysine), 59 (lysine), 86 (lysine), 87
(alanine), 89 (glutamine), 91 (serine), 93 (lysine), 95
(serine), 106 (threonine), 110 (serine), 136 (threonine), and
137 (leucine) of the cM3O heavy chain ented by SEQ ID
NO: 63ijlthe{SequenceIListingvdjjiglutamine, , e,
valine, lysine, serine, , arginine, alanine, arginine,
, isoleucine, alanine, glutamic acid, threonine,
arginine, threonine, leucine, and valine, respectively, was
named “M30—H2—type heavy chain”.
The amino acid sequence of the M30—H2—type heavy chain
is represented by SEQ ID NO: 87.
FP1214S 154 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
9)—2—3 M30—H3—type heavy chain:
A humanized M30 heavy chain designed by substituting
amirm>acid numbers 24 (glutamine), 28 (proline), 3O ne),
31 (valine), 35 (alanine), 39 (methionine), 59 (lysine), 89
(glutamine), 91 (serine), 93 (lysine), 95 (serine), 106
(threonine), 110 (serine), 136 (threonine), and 137 (leucine)
of the cM3O heavy chain represented by SEQ ID NO: 63 in the
SequenceIListingvnjjlvaline, alanine, valine, lysine, serine,
valine, alanine, isoleucine, alanine, ic acid,
threonine, arginine, threonine, leucine, and valine,
respectively, was named “M30—H3—type heavy chain”.
The amino acid sequence of the M30-H3-type heavy chain
is ented by SEQ ID NO: 89.
9)—2-4 M30—H4—type heavy chain:
A humanized M30 heavy chain ed by substituting
amino acidrnnnbers 24 (glutamine), 28 (proline), 3O (leucine),
31 (valine), 35 (alanine), 39 (methionine), 59 (lysine), 95
(serine), 106 (threonine), 110 (serine), 136 (threonine), and
137 (leucine) of the cM3O heavy chain represented by SEQ ID
NO: 63 in the Sequence Listing with valine, e, valine,
lysine, serine, valine, alanine, threonine, arginine,
threonine, leucine, and , respectively, was named
“M30-H4—type heavy chain”.
S 155WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
The amino acid sequence of the M30—H4—type heavy chain
is ented by SEQ ID NO: 91.
9)—3 Humanization of M30 light chain
9)—3—1 M30—Ll—type light chain:
A humanized M30 light chain designed by substituting
aminoacidnumberle(glutamine),25(serine),29(threonine),
(isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41
(methionine), 42 (threonine), 61 (serine), 62 (serine), 64
(lysine), 65 (proline), 66 (tryptophan), 77 (valine), 89
(serine),90 (tyrosine), 91(serine), ine), 99(alanine),
102 (alanine), 104 nine), 119 (threonine), 123 (leucine),
and 125 (leucine) of the cM30 light chain represented by SEQ
IDNO:59:UitheSequenceListingwithglutamicacid,threonine,
alanine, threonine, leucine, arginine, alanine, leucine,
, glutamine, alanine, arginine, leucine, leucine,
isoleucine,asparticacid,phenylalanine,threonine,leucine,
proline, phenylalanine, valine, glutamine, valine, and
isoleucine, respectively, was named 1—type light
chain”.
The amino acid sequence of the M30—L1—type light chain
is represented by SEQ ID NO: 71.
9)—3—2 M30—L2—type light chain:
A humanized M30 light chain designed by tuting
FP1214s 156WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
cidnumbers2l(glutamine),25(serine),29(threonine),
(isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41
(methionine), 42 (threonine), 61 (serine), 62 (serine), 64
(lysine), 65 (proline), 77 (valine), 89 (serine), 91 (serine),
97 (valine), 99 (alanine), 102 (alanine), 104 (threonine), 119
(threonine), 123 (leucine), and.125 (leucine) oftfluacM3O light
chain represented by SEQ ID NO: 59 in the ce Listing with
glutamic acid, ine, alanine, threonine, leucine,
arginine, alanine, leucine, serine, glutamine, alanine,
arginine, leucine, isoleucine, aspartic acid, threonine,
leucine, proline, phenylalanine, valine, glutamine, valine,
and isoleucine, respectively, was named “M30—L2—type light
chain”.
The amino acid sequence of the M30—L2—type light chain
is ented by SEQ ID NO: 73.
9)—3—3 M30-L3—type light chain:
A humanized M30 light chain ed by substituting
amino acid numbers 29 (threonine), 30 (isoleucine), 33
(alanine), 38 e), 39 (valine), 41 onine), 62
(serine), 65 (proline), 77 e), 91 (serine), 97 (valine),
99 (alanine), 102 (alanine), 104 nine), 119 (threonine),
123 (leucine), and 125 (leucine) of the cM30 light chain
represented by SEQ ID NO: 59 in the Sequence Listing with
alanine, threonine, leucine, arginine, alanine, leucine,
FP1214S 157 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
alanine, leucine, isoleucine, threonine, e, proline,
phenylalanine, valine, glutamine, valine, and isoleucine,
respectively, was named 3—type light chain”.
The amino acid sequence of the M30—L3-type light chain
is ented by SEQ ID NO: 75.
9)—3-4 M30-L4—type light chain:
A humanized M30 light chain designed by substituting
aminoacidnumberle(glutamine),25(serine),29(threonine),
3O (isoleucine), 33 ne), 38 e), 39 (valine), 41
(methionine), 42 (threonine), 61 (serine), 62 (serine), 64
(lysine), 66 (tryptophan), 77 (valine), 89 (serine), 9O
(tyrosine), 91 (serine), 96 (arginine), 97 (valine), 99
(alanine), 102 (alanine), 104 (threonine), 119 (threonine),
123 (leucine), and 125 (leucine) of the cM3O light chain
represented by SEQ ID NO: 59 in the Sequence Listing with
glutamic acid, threonine, alanine, threonine, leucine,
arginine, e, leucine, serine, glutamine, alanine,
ne, leucine, isoleucine, aspartic acid, phenylalanine,
threonine, serine, leucine, proline, phenylalanine, valine,
glutamine, valine, and isoleucine, respectively, was named
“M30—L4—type light chain”.
The amino acid sequence of the M30—L4-type light chain
is represented by SEQ ID NO: 77.
FP1214S 158 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
9)—3—5 M30—L5—type light chain:
A humanized M30 light chain designed by substituting
amino acid numbers 29 nine), 3O (isoleucine), 33
(alanine), 38 (lysine), 39 (valine), 41 (methionine), 62
(serine), 77 (valine), 91 (serine), 97 (valine), 99 (alanine),
102(alanine),104(threonine),119(threonine),123(leucine),
and 125 (leucine) of the cM3O light chain represented by SEQ
ID NO: 59 in the Sequence Listing with alanine, threonine,
e, arginine, alanine, e, alanine, isoleucine,
threonine,leucine,proline,phenylalanine,valine,glutamine,
valine, and isoleucine, respectively, was named “M30—L5—type
light chain”.
The amino acid sequence of the M30-L5—type light chain
is represented by SEQ ID NO: 79.
9)—3—6 M30—L6—type light chain:
A humanized M30 light chain designed by substituting
cidnumbers2l(glutamine),25(serine),29(threonine),
3O (isoleucine), 33 (alanine), 38 (lysine), 39 e), 41
(methionine), 42 (threonine), 61 (serine), 62 (serine), 64
(lysine), 66 (tryptophan), 77 (valine), 89 (serine), 9O
(tyrosine), 91 e), 97 (valine), 99 (alanine), 102
(alanine), 104 (threonine), 119 (threonine), 123 (leucine),
and 125 (leucine) of the cM3O light chain represented by SEQ
9:UitheSequenceListingwithglutamicacid,threonine,
FP1214s ngWGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
alanine, threonine, leucine, arginine, alanine, leucine,
serine, ine, alanine, arginine, leucine, cine,
aspartic acid, phenylalanine, threonine, leucine, proline,
phenylalanine, valine, glutamine, valine, and isoleucine,
respectively, was named “M30—L6—type light chain”.
The amino acid sequence of the M30—L6etype light chain
is represented by SEQ ID NO: 81.
9)—3—7 M30-L7—type light chain:
A humanized M30 light chain designed by tuting
aminoacidnumberle(glutamine),25(serine),29(threonine),
3O (isoleucine), 33 ne), 38 (lysine), 39 (valine), 41
(methionine), 42 (threonine), 61 (serine), 62 (serine), 64
(lysine), 66 (tryptophan), 77 (valine), 89 (serine), 91
(serine), 97 (valine), 99 (alanine), 102 (alanine), 104
(threonine), 119 (threonine), 123 (leucine), and 125 ne)
of the cM30 light chain represented by SEQ ID NO: 59 in the
Sequence g with glutamic acid, threonine, alanine,
threonine, leucine, arginine, alanine, e, serine,
glutamine, alanine, arginine, leucine, isoleucine, aspartic
acid, threonine, leucine, proline, alanine, valine,
glutamine, valine, and isoleucine, respectively, was named
“M30-L7—type light chain”.
The amino acid sequence of the M30—L7—type light chain
is represented by SEQ ID NO: 83.
FP1214s 16OWGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
(Example 10) tion of humanized antibody
)—l Construction.of M30—L1, M30—L2, , M30-L4, MBO—LS,
M30-L6, and —type light chain expression vectors
mprisingwageneencodinganiMBO-Ll,M30—L2,M30—L3,
M30—L4, M30—L5, M30—L6, or M30—L7-type light chain variable
region represented by amino acid numbers 21 to 128 in SEQ ID
NO: 71, amino acid numbers 21 to 128 in SEQ ID NO: 73, amino
acid numbers 21 to 128 in SEQ ID NO: 75, amino acid numbers
21 to 128 in SEQ ID NO: 77, amino acid numbers 21 to 128 in
SEQ ID NO: 79, amino acid numbers 21 to 128 in SEQ ID NO: 81,
or amino acid s 21 to 128 in SEQ IDIM3283 in the Sequence
Listing were synthesized (GENEART, Inc. Artificial Gene
Synthesis Service) based on SEQ ID NOS: 70, 72, 74, 76, 78,
80, andifiZaccordingtx>the nucleotide sequences corresponding
to the above SEQ ID NOS according to the amino acid sequences.
Then, each of the DNA fragments obtained by cleaving the
synthesized DNAs with the restriction enzymes NdeI and BsiWI
wasinsertedintotheuniversalchimericandhumanizedantibody
light chain expression vector (pEF6KCL) at the site cleaved
with the restriction enzymes NdeI and BsiWI, whereby MBO—Ll,
M30—L2, M30—L3, M30—L4, M30—L5, M30-L6, andIMBO—L7—type light
chain expression vectors were constructed.
The thus obtained expression s were named
CL/M30—Ll”, “pEF6KCL/M30—L2”, “pEF6KCL/M30-L3”,
FP1214s 161 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
“pEF6KCL/M30—L4”, “pEF6KCL/M30—L5”, CL/M30-L6”, and
“pEF6KCL/M30-L7”, respectively.
)—2 Construction.of M30—H1, M30—H2, M30—H3, andIMBO-H4—type
heavy chain expression vectors
DNAscomprisingaageneencodingen1M30—H1,M30—H2,M30—H3,
orM30—H4—typeheavychainvariableregionrepresenteflbyamino
acid numbers 20 to 141 in SEQ ID NO: 85, amino acid numbers
to 141 in SEQ ID NO: 87, amino acid numbers 20 to 141 in
SEQ ID NO: 89, or amino acid numbers 20 to 141 in SEQ ID NO:
91 in the Sequence Listing were synthesized (GENEART, Inc.
Artificial Gene Synthesis Service) based on SEQ IDDMM 84, 86,
88, amd90 according‘totfluanucleotide sequences corresponding
to the above SEQ ID NOS according to the amino acid ces.
Then, each of the DNA fragments obtained by ng the
synthesized DNAs with the ction enzyme BlpI was inserted
into the universal humanized dy heavy chain sion
vector (pEFlFCCU) at the site cleaved with the restriction
enzyme BlpI, whereby M30—H1, M30—H2, M30~H3, and M30—H4~type
heavy chain expression vectors were constructed.
The thus obtained expression vectors were named
“pEFlFCCU/M30—H1”,“pEFlFCCU/M30—H2”,“pEF1FCCU/M30-H3”,and
“pEFlFCCU/M30—H4”, respectively.
)—3 Production of humanized antibody
FP1214s 162 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
1.2 x 109 cells of FreeStyle 293F cells (Invitrogen
ation) in the thmic growth phase were seeded into
1.2 L of fresh FreeStyle 293 Expression Medium (Invitrogen
Corporation) and cultured for 1 hour by shaking at 90 rpm at
37°C in an 8% C02 incubator. 3.6 mg of polyethyleneimine
(Polyscience #24765) was dissolved in 20 ml of Opti—Pro SFM
medium(Invitrogeanrporation). Subsequently,eaheavychain
expression vector (0.4 mg) andealight chain.expression.vector
g);preparedxwith PureLink:HiPure Plasmid.Kit (Invitrogen
Corporation) were ded in 20 ml of Opti—Pro SFM medium.
Then, 20 ml of the obtained expression vectors/Opti—Pro SFM
mixed liquid was added to 20 ml of the obtained
polyethyleneimine/Opti—Pro SFM mixed liquid, and the
resultingmixturewasgentlystirredandthenleftforfiminutes.
Thereafter, thelnixture was added to the FreeStyle 293E‘cells,
and shaking culture at 90 rpm was performed for 7 days at 37°C
in an 8%(Xh incubator. The resulting culture atant was
filtered through a disposable capsule filter (Advantec
#CCS—O45—E1H).
A humanized dy of M30 obtained by a combination
of pEFlFCCU/M30—Hl and pEF6KCL/M30—Ll was named “M30—Hl—Ll”,
a humanized antibody of M30 obtained by a combination of
pEFlFCCU/MBO-Hl and pEF6KCL/M30—L2 was named “M30—H1—L2”, a
humanized. antibody' of M30 obtained tux a combination. of
pEFlFCCU/M30—H1 and pEF6KCL/M30—L3 was named “M30—Hl—L3”, a
FP1214S 163WGA/PN810023/Eng trans of PCT 6.9.13
4426167—1—WARENDS
humajuzed. antibody of M30 obtained. by' a combination of
pEFlFCCU/M30—Hl and pEF6KCL/M30—L4 was named “M30—Hl-L4", a
humaxuzed. antibody of M30 obtained by a combination. of
pEFlFCCU/MBO—H4 and pEF6KCL/M30-Ll was named “M30—H4—L1", a
humanized antibody of M30 obtained by a combination of
CU/MBO—H4 and pEF6KCL/M30—L2 was named “M30—H4—L2”, a
humanized. antibody’ of M30 obtained km! a combination. of
pEFlFCCU/M30—H4 and pEF6KCL/M30—L3 was named 4—L3”, a
humanized. antibody" of M30 ed tar a combination. of
pEFlFCCU/MBO—H4 and pEFGKCL/MBO—L4 was named “M30—H4—L4”, a
humanized. antibody' of M30 obtained knr a combination. of
pEFlFCCU/MBO-Hl and pEF6KCL/M30-L5 was named l-L5”, a
humanized. antibody‘ of M30 obtained by a combination. of
jpEFlFCCU/MBO—Pfi.andngF6KCL/M30—L6 was named “M30—Hl—L6”, and
a humanized antibody of M30 obtained by a combination of
CU/M30—Hl and pEF6KCL/M30—L7 was named “M30—H1—L7".
lO)—4 Purification of humanized antibody
The e supernatant obtained in Example lO)—3 was
purified by a ep process including rProtein.A affinity
chromatography (at 4 to 6°C) and ceramic hydroxyapatite (at
room temperature). A buffer exchange step after the
purification by in.A affinity chromatography and after
the purification by ceramic hydroxyapatite was performed at
room temperature.
First, 1100 to 1200 ml of the culture supernatant was
applied to MabSelect SuRe (manufactured by GE Healthcare
Bio—Sciences Co., Ltd., two HiTrap columns (volume: 1 ml)
connected.in series) equilibratedxvith PBS. After all culture
solution was poured.into the column, the colunwlwas washedxvith
to 3OImLof PBS. Subsequently, elution_was performed with
a ZIMarginine hydrochloride solution (pH 4.0), andaafraction
containing the dy was collected. The fraction was
applied to a desalting column (manufactured by GE care
Bio—Sciences Co., Ltd., two HiTrap desalting columns (volume:
ml) connected in series), whereby the buffer was exchanged
wittla buffer containing SImdsodiuanhosphate, SOIM4MES, and
mM NaCl at pH 6.5.
Further, the antibody solution subjected to buffer
ge was appliedixna.ceramic:hydroxyapatite<:olumn (Japan
Bio—Rad Laboratories, Inc., Bio—Scale CHTZ—l yapatite
column (volume: 2 ml)) brated with a buffer containing
mM NaPi, 50 mM MES, and 20 mM NaCl at pH 6.5. Then, linear
concentration. nt n with sodium. chloride was
med, and a fraction containing the antibody was
collected.
The fraction was applied to a desalting column
(manufactured by GE Healthcare Bio-Sciences Co., Ltd., two
HiTrap desalting columns (volume: 5 ml) connected in series),
whereby the liquid was exchanged with CBS (containing 10 mM
FP1214S 165WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
citrate buffer and 140 mM sodium chloride, pH 6.0).
Finally, the resulting solution was concentrated using
CentrifugalLN?FilterDeviceVIVASPDflZO(fractionalnwlecular
weight: 30 K, ius Co., Ltd., at 4°C), and the
concentration of IgG was adjusted to 1.0 mg/ml or more, and
the thus obtained solution was used as a purified sample.
lO)—5 g property of humanized antibody to B7-H3 antigen
The binding property of the humanized M30 antibody to
human B7—H3 which is an antigen was measured by a surface
plasmon.resonance (SPR) device (Biacore, Inc.). According to
comnwn procedures, an anti—human IgG antibody was immobilized
on a sensor chip, and then, each of the purified s of
the humanized M30 dies obtained in the above lO)-4 was
bound thereto. Thereafter, an extracellular domain
polypeptide of the B7—H3 t 2 antigen (manufactured by
R&D Systems, Inc., #2318—B3-050/CF) was added thereto at
different concentrations, and.the amount of the antigen.bound
to the antibody in a running buffer (phosphate , 0.05%
SP20)wasneasuredovertime. Theneasuredamountwasanalyzed
with dedicated software (BIAevaluation) and a dissociation
constant (Kd [M]) was calculated. The measurement was
performed using the cM30 antibody as a positive control for
eachuneasurement operation. The results are as shown in‘Table
3. All of the humanized M30 antibodies had binding activity
S 166WGA/PN810023/Eng trans of PCT spec/26.9.13
AA’)R1 67-1 -WARFNDS
against the B7—H3 antigen.
[Table 3]
KdW]
M30-H1-L1 8.7E-O8
M30-H1-L2 1.0E-O7
M30-H1—L3 1.0E—O7
M30-H1—L4 1.6E-O8
M30-H4-L1 1 .3E-07
M30-H4-L2 1 .4E-07
M30-H4-L3 1 .6E-07
M30-H4-L4 1 .2E-O8
M30-H1-L5 1.9E—09
M30-H1-L6 3.4E-09
M30-H1-L7 9
cM30 73E40
Example 11. Measurement of competitive tory activities
of cM30 antibody and humanized M30 antibody against M30
antibody for the binding to B7—H3 antigen
The competitive inhibitory activities of the cM30
antibody and the zed M30 antibody l—L4 antibody)
against the M30 antibody for the binding to the B7—H3 variant
1 and variant 2 were measured by the following method.
By using EZ—Link Sulfo—NHS—LC Biotinylation Kit
actured by Thermo Scientific Corporation, #21435) and
according to the attached protocol, the respective mouse
monoclonal antibodies M30 were biotinylated nafter the
respective biotinylated M30 antibodies were referred to as
“bM30”) . Further, as a buffer to be used in the following ELISA
S 167 WGA/PN810023/Eng trans of PCT spec/26.9.13
4476167—1-WARENDS
method, BD OPTI EIA (BD ences, Inc., #550536) was used
in all cases.
Each of the extracellular domain polypeptide of the B7—H3
variant 1 (manufactured by R&D Systems, Inc. , #l949—B3-050/CF)
and the extracellular domain ptide of the B7—H3 variant
2 (manufactured by R&D Systems, Inc., #2318—83—050/CF) was
d to 0.5 ug/ml with a coating buffer and the resulting
solution was dispensed at 100 uL/well in an immunoplate
(manufactured by Nunc, lnc., #442404) . Then, the plate was
left to stand overnight at 4°C, whereby the protein was adsorbed
to the plate. On the next day, an assay diluent was dispensed
at 200 uL/well, and the plate was left to stand at room
ature for 4 hours.
After the solution in each well was removed, a mixed
solution of the biotinylated antibody at 5 ug/ml and an
unlabeled antibody at each concentration (0 ug/ml, l ug/ml,
Sug/ml, 25 ug/ml, 50 ug/ml, or 125 ug/ml) was dispensed at 100
uL/well in an assay diluent, and the plate was left to stand
at room temperature for 1 hour.
After each well was washed twice with a wash buffer, a
streptavidin—horseradish peroxidase conjugate (manufactured
diluted
by GE care Bio-Sciences Corporation, 31V)
to 500—fold with an assay diluent was added at 100 uL/well,
and.the plate was left to stand at room temperature for 1 hour.
After the solution in each well was removed and each well
FP1214s 168 WGA/PN810023/Eng trans of PCT spec/26.9.13
4476167-1—WARENDS
was washed twice with a wash buffer, a substrate solution was
added at 100 uL/well, and a color pment reaction was
dtoproceedwhilestirringthereactionndxture. After
completion of the color development, a blocking buffer was
added thereto at 100 ul/well to stop the color development
reaction. Then, an absorbance at 450 nm was measured using
a plate reader.
As a result, the ance of the well to which only
bM30wasaddedwas2.36:t0.05(meanistandarddeviation(n=12))
in the plate to which the polypeptide of the extracellular
domain of the B7—H3 variant 1 was attached, and 1.90 i 0.20
(mean i standard deviation (n=12)) in the plate to which the
polypeptide of the extracellular domain of the B7—H3 t
2 was attached.
The absorbances in the graphs of Fig. 10 are each
expressed as a mean i standard deviation (n=3). The control
IgG did not inhibit the binding of bM30 to B7-H3.
On the other hand, it was shown that the binding of bM30
to B7—H3 is inhibited by the M30 antibody , the cM30
antibody, which is a chimeric antibody of the M30 antibody,
and the M30—Hl-L4, which is a humanized antibody, in both of
the plate to which the polypeptide of the extracellular domain
of the B7—H3 variant 1 was attached and the plate to which the
FP1214S 169 WGA/PN810023/Eng trans of PCT spec/26.9.13
4478167-1—WARENDS
polypeptide of the extracellular domain of the B7—H3 variant 2
was attached.
[019 3]
That is, it was shown that the cM30 antibody and the
humanized antibody (M30—H1~L4 antibody) recognize the same
epitope of the B7—H3 antigen as the M30 antibody.
e 12. Activity of humanized M30 antibody
12)—1 ADCP activity of humanized M30 antibody
PBMCsofeahealthysubjectwereisolatedaccordingtx>common
procedures and.suspended.in.10% FBS~containingfiRPMI 1640 and then
seeded in a flask. The cells were cultured overnight in a C02
incubator. The culture supernatant.was removed, andtx>the cells
attached.to the flask, 10% FBS—containing RPMI 1640 mented
-CSF and GM—CSF (PeproTech, Inc.) was added, and the cells
were cultured for 2 weeks. The cells were detached with TrprE
and collected. Then, the cells were added to a 24-well plate at
500 ul/well (1 x 105cells/well) and cultured overnight at 37°C.
The thus prepared cells were used as or cells.
The labeling of NCI—H322 cells to be used as target cells
was performed using PKH26 dye labeling kit (Sigma Co., Ltd.).
The target cells were detached.with TrprE shed twice with
PBS. The cells were suspended in Diluent C at 1 x 107 ml.
PKH26 dye stock (1 mM) was diluted to 8 uM with Diluent C, and
immediately thereafter, the diluted dye solution was added
thereto in.an.amount equal to that of the cell suspension. The
resulting e was left at room ature for 5 minutes.
Then, 1 ml of serum was added thereto, and further, a medium
with.serun1was added thereto, and washing was performed.twice.
Each of the M30 antibody, the cM3O antibody, and the
humanized.M30 antibody (M30—H1—L4 antibody) was diluted to 20
ug/ml with 10% FEB—containing RPMI 1640 (Invitrogen
Corporation). uently, the target cells (NCI—H322
cells) were dispensed at 2 x 106 cells/100 ul/tube and mixed.
The resulting mixture was left to on ice for utes.
The supernatant was removed, and the cells were washed twice
with a culture solution and suspended in 500 pl of a culture
solution.
The supernatant was removed.fron1the effector cells, and
the cells having been treated with the M30 antibody, the cM3O
antibody, or the humanized M30 antibody (M30~H1~L4 antibody)
and suspended in the culture solution were added thereto and
mixed therewith. Then, the cells were cultured for 3 hours
in a C02 incubator.
Thereafter, the cells were detached with Trypsin—EDTA
and collected.
To the collected cells ouse CDllb
, an FITC-labeled
antibody (Becton, Dickinson.and Company, Ltd.) was added, and
the resulting e was left to stand.on ice for 30 minutes.
The supernatant was removed, and the cells were washed
twice with a culture solution.
The collected cells were ded in 300 pl of a culture
medium and analyzed by FACS Calibur (Becton, Dickinson and
Company, Ltd.). In the CDllb-positive macrophages, a
PKH26—positive fraction was evaluated as
phagocytosis—positive cells.
The results are shown in Fig. 11.
In the group with the addition of the zed M30
antibody (M30—H1—L4 antibody), ADCP activity against the
NCI—H322 cells was observed.in.the anner as in.the groups
with the addition of the M30 antibody or the cM30 antibody.
12)—2 ADCC activity of humanized M30 antibody
PBMCs of a healthy subject were isolated according to
commonproceduresandsuspendedin10%FBS-containingRPMI1640
and.then.seeded.inaiflask. The cells were cultured.overnight
in a C02 incubator.
The floating cells were ted, followedlnra washing
procedure, and the resulting cells were used as peripheral
blood lymphocytes (PBLs). The obtained PBLs were ded
in phenol red—free RPMI 1640 (manufactured by Invitrogen
ation) containing 10% Fetal Bovine Serum (manufactured
by Invitrogen Corporation) (hereinafter abbreviated.as “ADCC
mediu "), and the cell suspension was passed through a cell
strainer (pore size: 40 um, manufactured by BD Biosciences,
Ltd.). Then, the viable cells were d by a trypan blue
dye exclusion assay. After the PBLs suspension was
centrifuged, the medium was removed, and the cells were
resuspended in the.ADCC1nediun1at a viable cell density of 2.5
x 106 cells/ml and used as effector cells.
NCI—H322cellsweretreatedwithtrypsin,andthetreated
cells were washed with 10% FBS—containing RPMI 1640 and then
resuspended in 10% PBS—containing RPMI 1640. The cells (4 X
106 cells) were mixed with chromium—51 (5550 kBq) sterilized
through a O for 1 hour
. 22 um , and labeling was med
under the conditions of 37°C and 5% C02. The labeled cells were
washed three times with the ADCC medium, and the cells were
resuspended at 2 X 105 cells/ml in the ADCC medium and used
as target cells.
The target cells at a cell density of 2 x 105 cells/ml
were dispensed at 50 ul/well in a l U—shaped bottom
microplate. 50 ul of each of the cM30 antibody and the
humanized M30 antibodies 1—L4, M30—H4—L4, M30—H1—L5,
M30—H1—L6, and M30—H1—L7) was added.thereto, diluted.with the
ADCC medium so that the final concentration of the antibody
after adding the effector cells was 1, 10, 100, or 1000 ng/ml.
Then, 100 pl of the effector cells at a cell density of 2.5
x 106cells/ml were added thereto, and.the cells were cultured
for 4 hours under the conditions of 37°C and 5% C02. The
supernatant was collected in a LumaPlate (manufactured by
PerkinElmer, Inc.), and.gamma radiation emitted.therefron1was
ed using a gamma counter. The percentage of cell lysis
caused by the ADCC activity was calculated according to the
following equation.
Percentage of cell lysis ( o\° ) = (A—B) / (C—B) x 100
A: Radiation count from the sample well
B: Average spontaneous radiation emission count (from
wells to which the dy and the effector cells were not
added) (n=3). The same procedure as that for the sample well
wasperformedexceptthattfluaADCCHQdimnwasaddedjjlanamount
of 50 pl at the time of adding the antibody and in an amount
of 100 pl at the time of adding the effector cells.
C: Average maximum.radiation emission count (fronlwells
in which the target cells were dissolved with a surfactant)
(n=3). The same procedure as that for the sample well was
performed except that 50 pl of the ADCC medium was added at
the time of adding the antibody and 100 pl of the ADCC medium
containing 2% (v/v) Triton X—100 was added at the time of adding
the or cells.
FP1214S 174 WGA/PN810023/Eng trans of PCT 6.9.13
4426167—1—WARENDS
The results are shown in Table 4 and Fig. 12.
Thedatashownareentaverageoftriplicateneasurements,
and.the error bars represent rd deviations. The P value
was calculated using Student’s t—test.
In the group with the addition of the M30—H1—L4 antibody,
ADCC activity was observed in the same manner as in the group
with the addition of the cM30 antibody. Also other humanized
M30 antibodies (M30—H4—L4, M30—H1-L5, M30—H1—L6, and
M30—H1—L7) were observed to have ADCC activity in the same
manner.
[Table 4]
1 ng/mL 10 ng/mL 100 ng/mL 1000 ng/mL
Average Standard e Standard Average rd Average Standard
(%) deviation (%) deviation (%) deviation (%) deviation
human '96 -_m 53’m 0-5
01430 882 4.0
M30-H1-L4 0.1 83.6 0.7 84.7 1.0
888888 8.8 88.8 8.8 88.8 8.8
888-8818“- 8.8 88.8 8.8 88.8 8.8
M30«H1—L6 n__ 2.2 74.7 4.0 81.3 2.0
M30—H1-L7 0.7 71.7 2.0 80.7 0.7
12)—3 In vivo antitumor effect of humanized M30 antibody
MDA—MB-231 cells were detached from a culture flask by
a trypsin treatment, and then suspended in 10% PBS—containing
RPMI 1640 medium (Life Technologies Corporation), followedioy
centrifugation and the supernatant was d. The cells
FP1214S 175WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
were washed twice with the same , and then suspended in
BD el Basement Membrane Matrix (manufactured by BD
Biosciences, Inc.). Then, the cells were implanted
subcutaneously in the axillary region of each of a group of
mice (FOX CHASE SCID /Icr-scid/scidJcl, CLEA Japan,
Inc.) at 6 weeks of age at a dose of 5 x 106 cells/mouse. The
day of implantation was taken as day 0, and on days 14, 21,
28,35,and42,thehumanizedM30antibody(M30—Hl—L4antibody)
was intraperitoneally administered at a dose of 10, 1, 0.1,
or0.0lmg/kg(about200,20,2,<N:O.2ug/mouse,respectively).
The tumor volume was measured on days 14, 18, 21, 25, 28, 31,
, 39, 42, 45, and 49, and the antitumor effect of the
administration of the antibody was examined.
As a result, in the groups of administration of the
humanized.M30 antibody (M30—H1—L4 antibody) at 10, 1, and 0.1
mg/kg, the tumor growth was significantly suppressed as
compared with the untreated group in which the antibody was
not administered. In the groups of administration of the
humanized M30 antibody (M30—H1-L4 antibody) at 10, 1, and 0.1
mg/kg,thetumorgrowthinhibitionratio(2100-(averagetumor
weight in antibody administration group) / ge tumor
weight in untreated group) x 100) compared.with the untreated
group in terms of the tumor weight on day 49 was 67, 54, and
51%, respectively, and the P values were all P<0.000l. The
P values were calculated using Dunnett's le comparison
FP1214s l76WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167WARENDS
test.
r, the tumor growth inhibition ratio (%) (= 100
— (average tumor volume in antibody administration group) /
(average tumor volume in untreated group) x 100) of the
M30—Hl—L4 antibody on day 49 in the 10, l, 0.1, and 0.01 mg/kg
stration groups was 84, 68, 61, and 30%, respectively.
Accordingly, the humanized M30 antibody (M30—Hl-L4 antibody)
was observed to have a very strong antitumor effect in vivo
in.the same manner as the M30 antibody and the cM3O antibody,
and it was confirmed that the effect was exhibited in a
dose-response manner (Fig. 38).
Industrial Applicability
The anti—B7-H3 antibody of the ion has antitumor
activity, and a pharmaceutical composition comprising the
anti—B7-H3 antibody can be an anticancer agent.
Sequence Listing Free Text
SEQ ID NO: 1: PCR primer 1
SEQ ID NO: 2: PCR primer 2
SEQ ID NO: 3: CMV promoter primer: primer 3
SEQ ID NO: 4: BGH reverse primer: primer 4
SEQ ID NO: 5 cc nucleotide sequence of B7-H3 variant 1
FP1214s 177 WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
SEQ ID NO: 6: amino acid sequence of B7—H3 variant 1
SEQ ID NO: 7: PCR primer 5
SEQ ID NO: 8: PCR primer 6
SEQ ID NO: 9: nucleotide sequence of B7—H3 variant 2
SEQ ID NO: 10: amino acid ce of B7-H3 variant 2
SEQ ID NO: 11: PCR primer 7
SEQ ID NO: 12: PCR primer 8
SEQ ID NO: 13: PCR primer 9
SEQ ID NO: 14: PCR primer 10
SEQ ID NO: 15: PCR primer 11
SEQ ID NO: 16: PCR primer 12
SEQ ID NO: 17: PCR primer 13
SEQ ID NO: 18: PCR primer 14
SEQ ID NO: 19: PCR primer 15
SEQ ID NO: 20: nucleotide sequence of B7—H3 Ing
SEQ ID NO: 21: amino acid sequence of B7—H3 Ing
SEQ ID NO: 22: nucleotide sequence of B7—H3 IgCl
SEQ ID NO: 23: amino acid sequence of B7~H3 IgCl
SEQ ID NO: 24: nucleotide sequence of B7—H3 IgV2
SEQ ID NO: 25: amino acid sequence of B7—H3 IgV2
SEQ ID NO: 26: nucleotide sequence of B7—H3 IgC2
SEQ ID NO: 27: amino acid sequence of B7—H3 IgC2
SEQ ID NO: 28: nucleotide sequence of B7—H3 IgCl—V2-C2
SEQ ID NO: 29: amino acid sequence of B7-H3 IgCl—V2*C2
SEQ ID NO: 30: nucleotide ce of B7—H3 IgV2—C2
FP1214s 1j78 810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
SEQ ID NO: 31: amino acid sequence of B7—H3 2
SEQ ID NO: 32: nucleotide sequence of B7RP—l
SEQ ID NO: 33: amino acid sequence of B7RP—l
SEQ ID NO: 34: nucleotide sequence of B7—Hl
SEQ ID NO: 35: amino acid sequence of B7—Hl
SEQ ID NO: 36: nucleotide sequence of B7-DC
SEQ ID NO: 37: amino acid sequence of B7—DC
SEQ ID NO: 38: nucleotide sequence of CD80
SEQ ID NO: 39: amino acid sequence of CD80
SEQ ID NO: 40: nucleotide sequence of CD86
SEQ ID NO: 41: amino acid ce of CD86
SEQ ID NO: 42: nucleotide sequence of B7-H4
SEQ ID NO: 43: amino acid sequence of B7—H4
SEQIHDNO:44:Neterminalaminoacidsequenoeofnwuseantibody
M30 heavy chain
SEQIU)NO:45:N-terminalaminoacidsequenceofnmmseantibody
M30 light chain
SEQ ID NO: 46: PCR primer l6
SEQ ID NO: 47: PCR primer l7
SEQ ID NO: 48: PCR primer l8
SEQ ID NO: 49: PCR primer l9
SEQIDNO:50:nucleotidesequenceochNAencodingM30antibody
heavy chain
SEQ ID NO: 51: amino acid sequence of M30 dy heavy chain
SEQIDNO:52:nucleotidesequenceofcDNAencodingM30antibody
FP1214s 179WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167—1-WARENDS
light chain
SEQ ID NO: 53: amino acid sequence of M30 antibody light chain
SEQ ID NO: 54: PCR primer 20
SEQ ID NO: 55: PCR primer 21
SEQ ID NO: 56: DNA sequence encoding human K chain secretory
signal, human K chain constant , and human poly~A
additional signal
SEQ ID NO: 57: DNA fragment comprising DNA sequence encoding
amino acids of signal sequence and constant region of human
IgG1
SEQIDNO:58:nucleotidesequenceochNAencodingM30antibody
chimera—type light chain
SEQ ID NO: 59: amino acid sequence of M30 antibody chimera-type
light chain
SEQ ID NO: 60: PCR primer 22
SEQ ID NO: 61: PCR primer 23
SEQIDNO:62:nucleotidesequenceochNAencodingM30antibody
chimera-type heavy chain
SEQIDPMM 63:amino acid.sequence ofIMBO antibody chimera—type
heavy chain
SEQ ID NO: 64: PCR primer 24
SEQ ID NO: 65: PCR primer 25
SEQ ID NO: 66: PCR primer 26
SEQ ID NO: 67: PCR primer 27
SEQ ID NO: 68: PCR primer 28
180WGA/PN810023/Eng trans of PCT 6.9.13 FP1214S
4426167-1—WARENDS
SEQ ID NO: 69: PCR primer 29
SEQ ID NO: 70: nucleotide sequence of MBO—Ll—type light chain
SEQ ID NO: 71: amino acid sequence of M30—Ll—type light chain
SEQ ID NO: 72: nucleotide sequence of —type light chain
SEQ ID NO: 73: amino acid sequence of M30—L2—type light chain
SEQ ID NO: 74: nucleotide sequence of M30~L3-type light chain
SEQ ID NO: 75: amino acid sequence of M30—L3—type light chain
SEQ ID NO: 76: nucleotide sequence of M30—L4~type light chain
SEQ ID NO: 77: amino acid sequence of M30—L4—type light chain
SEQ ID NO: 78: nucleotide sequence of M30—L5—type light chain
SEQ ID NO: 79: amino acid sequence of MBO-LS—type light chain
SEQ ID NO: 80: tide ce of M30—L6—type light chain
SEQ ID NO: 81: amino acid sequence of M30-L6-type light chain
SEQ ID NO: 82: nucleotide sequence of M30—L7—type light chain
SEQ ID NO: 83: amino acid sequence of M30—L7—type light chain
SEQ ID NO: 84: nucleotide sequence of M30—H1—type heavy chain
SEQ ID NO: 85: amino acid sequence of M30-Hl—type heavy chain
SEQ ID NO: 86: nucleotide sequence of M30-H2-type heavy chain
SEQ ID NO: 87: amino acid sequence of MBO-HZ—type heavy chain
SEQ ID NO: 88: nucleotide sequence of M30—H3-type heavy chain
SEQ ID NO: 89: amino acid sequence of M30—H3-type heavy chain
SEQ ID NO: 90: tide ce of M30—H4—type heavy chain
SEQ ID NO: 91: amino acid ce of M30-H4—type heavy chain
SEQ ID NO: 92: amino acid sequence of M30 antibody CDRHl
SEQ ID NO: 93: amino acid sequence of M30 antibody CDRHZ
FPlZl4s 181 WGA/PNBIOOZB/Eng trans of PCT spec/26.9.13
4426167—1—WARENDS
SEQ ID NO: 94: amino acid sequence of M30 antibody CDRH3
SEQ ID NO: 95: amino acid sequence of M30 antibody CDRLl
SEQ ID NO: 96: amino acid sequence of M30 antibody CDRLZ
SEQ ID NO: 97: amino acid sequence of M30 dy CDRL3
FP1214S 182WGA/PN810023/Eng trans of PCT spec/26.9.13
4426167-1—WARENDS
Claims (41)
1. An antibody characterized by having the following properties of: (a) specifically binding to B7—H3; (b) having an dy—dependent cell—mediated phagocytosis (ADCP) ty; and (c) having an in vivo antitumor activity, or a functional fragment of the antibody, wherein said antibody or functional fragment thereof ses: CDRHl consisting of an amino acid sequence represented by SEQ ID NO: 92, CDRH2 consisting of an amino acid sequence represented by SEQ ID NO: 93, and CDRH3 consisting of an amino acid ce represented.by SEQ ID NO: 94 as complementarity determining regions of the heavy chain; and comprises CDRLl consisting of an amino acid sequence represented by SEQ ID NO: 95, CDRL2 consisting of an amino acid sequence represented.by SEQ ID NO: 96, and CDRL3 consisting of an amino acid sequence represented.by SEQ ID NO: 97 as complementarity determining regions of the light chain.
2. The antibody or a functional nt of the antibody according to claim 1, wherein B7—H3 is a molecule comprising an amino acid sequence represented by SEQ ID NO: 6 or 10.
3. The antibody or a functional fragment of the antibody according to claimILor 2, whichloinds to IgCl and/or IgC2 each of which is a domain of B7—H3.
4. The antibody or a onal fragment of the antibody according to Claim 3, wherein IgCl is a domain comprising an amino acid ce represented by amino acid numbers 140 to 244 in SEQ ID NO: 6, and IgC2 is a domain sing an amino acid sequence represented by amino acid numbers 358 to 456 in SEQ ID NO: 6 .
5. The antibody or a onal fragment of the antibody according to any one of claims 1 to 4, which has a competitive inhibitory activity against M30 antibody for the binding to B7—H3 .
6. The antibody or a functional fragment of the antibody according to any one of claims 1 to 5, which. has an antibody—dependent cellular‘ cytotoxicity LADCC) activity and/or a complement—dependent cytotoxicity (CDC) activity.
7. The antibody or a functional fragment of the antibody according to any one of claims 1 to 6, n the tumor is cancer.
8. The antibody or a functional fragment of the antibody according to claim 7, wherein the cancer is lung cancer, breast cancery te cancer; pancreatic cancer, colorectal cancer, a melanoma, liver cancer, n cancer, bladder cancer, stomach cancer, esophageal cancer, or kidney cancer.
9. The antibody or a functional fragment of the antibody according to any one of claims 1 to 8, which comprises a heavy chain variable region consisting of an amino acid ce represented by amino acid numbers 20 to 141 in SEQ ID NO: 51 and a light variable region.consisting of an amino acid sequence represented by amino acid numbers 23 to 130 in SEQ ID NO: 53 .
10. The antibody or a functional fragment of the antibody according to any one of claims 1 to 9, wherein a constant region is a human—derived constant region.
11. The antibody or a onal fragment of the antibody according to claim 10 , which comprises a heavy chain consisting of an amino acid ce represented by SEQ ID NO: 63 and a light chain ting of an amino acid sequence represented by SEQ ID NO: 59.
12. The antibody or a functional fragment of the antibody according to any one of claims 1 to 11, which is humanized.
13. The antibody or a functional fragment of the antibody according to clain112, which.comprises: a heavy chairlvariable region consisting of an amino acid ce selected from the group consisting of (a) an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 85, (b) an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 87, (c) an amino acid ce represented by amino acid numbers 20 to 141 in SEQ ID NO: 89, (d) an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 91, (e) an amino acid ce having a homology of at least 95% or more with any of the sequences (a) to (d), and (f) an amino acid sequence wherein.one or several amino acids are deleted, substituted or added in any of the sequences (a) to (d); and a light chain variable region consisting of an amino acid sequence selected from the group consisting of (g) an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 71, (h) an amino acid sequence ented by amino acid numbers 21 to 128 in SEQ ID NO: 73, (i) an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 75, (j) an amino acid sequence represented by amino acid s 21 to 128 in SEQ ID NO: 77, (k) an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 79, (1) an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 81, (m) an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 83, (n) an amino acid sequence having a gy of at least 95% or more with any of the sequences (g) to (m), and (o) an.amino acid sequence wherein one or several amino acids are deleted, substituted or added in any of the sequences (9) to (m).
14. The antibody or a functional fragment of the antibody according to claim 13, which comprises a heavy chain.variable region and a light chain variable region selected from the group consisting of: a heavy chain le region consisting of an amino acid sequence represented by amino acid s 20 to 141 in SEQ ID NO: 85 and a light chain variable region consisting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 71; a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 85 and a light chain variable region consisting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 73; a heavy chain variable region consisting of an amino acid sequence ented by amino acid numbers 20 to 141 in SEQ ID NO: 85 and a light chain le region consisting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 75; a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 85 and a light chain variable region consisting of an amino acid ce represented by amino acid numbers 21 to 128 in SEQ ID NO: 77; a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 85 and a light chain variable region consisting of an amino acid sequence represented by amino acid s 21 to 128 in SEQ ID NO: 79; a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 85 and a light chain variable region ting of an amino acid sequence ented by amino acid numbers 21 to 128 in SEQ ID NO: 81; a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 85 and a light chain variable region ting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 83; a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 91 and a light chain variable region consisting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 71; a heavy chain variable region consisting of an amino acid sequence represented by amino acid s 20 to 141 in SEQ ID NO: 91 and a light chain le region consisting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 73; a heavy chain le region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 91 and a light chain variable region consisting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 75; and a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 20 to 141 in SEQ ID NO: 91 and a light chain variable region consisting of an amino acid sequence represented by amino acid numbers 21 to 128 in SEQ ID NO: 77 .
15. The antibody or a functional fragment of the antibody according to claim 13 or 14, which comprises a heavy chain and a light chain selected from the group consisting of: a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light chainconsistingofauiaminoacidsequencerepresentaibyamino acid numbers 21 to 233 in SEQ ID NO: 71; a.heavy chain.consisting'of an.amino acid sequence represented by amino acid s 20 to 471 in SEQ ID NO: 85 and a light chain consisting of an amino acid ce represented by amino acid numbers 21 to 233 in SEQ ID NO: 73; a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by amino acid s 21 to 233 in SEQ ID NO: 75; a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light chain consisting of an amino acid sequence ented by amino acid numbers 21 to 233 in SEQ ID NO: 77; a heavy chain consisting of an amino acid sequenCe represented by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by amino acid numbers 21 to 233 in SEQ ID NO: 79; a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light chain consisting of an amino acid sequence ented by amino acid numbers 21 to 233 in SEQ ID NO: 81; a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by amino acid numbers 21 to 233 in SEQ ID NO: 83; a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 91 and a light chain ting of an amino acid ce represented by amino acid numbers 21 to 233 in SEQ ID NO: 71; a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 91 and a light chain consisting of an amino acid sequence represented by amino acid numbers 21 to 233 in SEQ ID NO: 73; a heavy chain consisting of an amino acid sequence represented by amino acid numbers 20 to 471 in SEQ ID NO: 91 and a light chain consisting of an amino acid ce represented by amino acid numbers 21 to 233 in SEQ ID NO: 75; and a heavy chain consisting of an amino acid sequence ented by amino acid numbers 20 to 471 in SEQ ID NO: 91 and a light chain consisting of an amino acid sequence represented by amino acid numbers 21 to 233 in SEQ ID NO: 77.
16. The antibody or a functional fragment of the antibody ing to any one of claims 13 to 15, which comprises a heavy chain and a light chain ed from the group consisting of: a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 71; a heavy chain ting of an amino acid sequence represented by SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 73; a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 75; a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 77; a heavy chain ting of an.amino acid sequence represented by SEQ ID NO: 85 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 79; a heavy'chain consisting of an.amino acid.sequence represented by SEQ ID NO: 85 and a light chain consisting of an amino acid ce represented by SEQ ID NO: 81; a heavy'chain.consisting of an.amint>acid.sequence represented by SEQ ID NO: 85 and a light Chain.consisting of an amino acid sequence represented by SEQ ID NO: 83; a heavy chain.consisting of an amino acid.sequence represented by SEQ ID NO: 91 and a light chain ting of an amino acid ce represented by SEQ ID NO: 71; a heavy chain consisting of an.amino acid sequence represented by SEQ ID NO: 91 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 73; a.heavy chain.consisting of an.aminc>acid.sequence represented by SEQ ID NO: 91 and a light chain consisting of an amino acid sequence ented by SEQ ID NO: 75; and a_heavy chain consisting ofau1amint>acid.sequence represented by SEQ ID NO: 91 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 77.
17. The functional fragment of the antibody according to any one of claims 1 to 16, n the functional fragment is selected from the group consisting of Fab, F(ab)2, Fab’ and
18. A.polynucleotide encoding the antibody or a functional fragment of the antibody according to any one of claims 1 to
19. The polynucleotide according to claim 18, which comprises a tide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 50 and a nucleotide sequence represented by tide numbers 67 to 390 in SEQ ID NO: 52.
20. The polynucleotide according to claim 18 or 19, which comprises a nucleotide ce represented by SEQ ID NO: 62 and a nucleotide sequence represented by SEQ ID NO: 58.
21. The polynucleotide according to claim 18 or 19, which comprises: a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence ented by nucleotide numbers 58 to 423 in SEQ ID NO: 84, (b) a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 86, (c) a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 88, (d) a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 90, and (e) a nucleotide sequence comprising a cleotide which hybridizes to a cleotide consisting of a nucleotide sequence complementary to any of the nucleotide sequences (a) to (d) under stringent conditions; and a nucleotide sequence selected from the group consisting (f) a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 70, (g) a nucleotide sequence represented by tide numbers 61 to 384 in SEQ ID NO: 72, (h) a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 74, (i) a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 76, (j) a nucleotide ce represented by nucleotide numbers 61 to 384 in SEQ ID NO: 78, (k) a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 80, (l) a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 82, and (m) a nucleotide sequence comprising a polynucleotide which hybridizes to a cleotide consisting of a nucleotide sequence mentary to any of the nucleotide sequences (f) to (1) under stringent conditions.
22. The polynucleotide according to claim 21, which comprises nucleotide sequences selected from the group ting of: a nucleotide ce represented.by nucleotide numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 70; a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide ce represented by nucleotide numbers 61 to 384 in SEQ ID NO: 72; a nucleotide sequence ented by nucleotide numbers 58 to 423 in SEQ ID NO: 84 and a tide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 74; a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 76; a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide sequence represented by tide s 61 to 384 in SEQ ID NO: 78; a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide sequence ented by nucleotide numbers 61 to 384 in SEQ ID NO: 80; a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 84 and a nucleotide ce represented by nucleotide numbers 61 to 384 in SEQ ID NO: 82; a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 90 and a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 70; a nucleotide sequence represented by nucleotide numbers 58 to 423 in SEQ ID NO: 90 and a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 72; a nucleotide sequence represented by nucleotide s 58 to 423 in SEQ ID NO: 90 and a nucleotide sequence represented by nucleotide s 61 to 384 in SEQ ID NO: 74; and a nucleotide ce represented.by nucleotide numbers 58 to 423 in SEQ ID NO: 90 and a nucleotide sequence represented by nucleotide numbers 61 to 384 in SEQ ID NO: 76.
23. The polynucleotide according to claim 21 or 22, which ses nucleotide sequences selected from the group consisting of: a nucleotide sequence represented by nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 70; a nucleotide sequence represented.by nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 72; a nucleotide sequence represented.by tide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 74; a nucleotide sequence represented by nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide ce represented by nucleotide numbers 61 to 699 in SEQ ID NO: 76; a nucleotide sequence ented by nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 78; a nucleotide sequence represented.by nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide s 61 to 699 in SEQ ID NO: 80; a nucleotide sequence ented.by'nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and a nucleotide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 82; a nucleotide sequence represented.by'nucleotide numbers 58 to 1413 in SEQ ID NO: 90 and a tide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 70; a nucleotide sequence represented.by nucleotide numbers 58 to 1413 in SEQ ID NO: 90 and a nucleotide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 72; a nucleotide sequence ented.by nucleotide numbers 58 to 1413 in SEQ ID NO: 90 and a nucleotide sequence represented by nucleotide s 61 to 699 in SEQ ID NO: 74; and a nucleotide sequence represented.by nucleotide numbers 58 to 1413 in SEQ ID NO: 90 and a tide sequence represented by nucleotide numbers 61 to 699 in SEQ ID NO: 76.
24. The polynucleotide according to any one of claims 21 to 23, which comprises nucleotide sequences selected from the group ting of: a nucleotide sequence represented by SEQ ID NO: 84 and a nucleotide sequence represented by SEQ ID NO: 70; a nucleotide sequence represented by SEQ ID NO: 84 and a nucleotide sequence represented by SEQ ID NO: 72; a tide sequence represented by SEQ ID NO: 84 and nucleotide sequence represented by SEQ ID NO: 74; a nucleotide sequence represented by SEQ ID NO: 84 and nucleotide sequence represented by SEQ ID NO: 76; a nucleotide sequence represented by SEQ ID NO: 84 and nucleotide sequence represented by SEQ ID NO: 78; a nucleotide sequence represented by SEQ ID NO: 84 and nucleotide sequence represented by SEQ ID NO: 80; a nucleotide sequence represented by SEQ ID NO: 84 and nucleotide sequence represented by SEQ ID NO: 82; a nucleotide sequence represented by SEQ ID NO: 90 and nucleotide sequence represented by SEQ ID NO: 70; a nucleotide sequence represented by SEQ ID NO: 9O and nucleotide sequence ented by SEQ ID NO: 72; a tide sequence represented by SEQ ID NO: 90 and nucleotide sequence represented by SEQ ID NO: 74; and a nucleotide sequence ented by SEQ ID NO: 90 and nucleotide ce represented by SEQ ID NO: 76.
25. An expression vector comprising a polynucleotide according to any one of claims 18 to 24.
26. A host cell, which is transformed with the sion vector according to claim 25, wherein the host cell is not a human cell in vivo.
27. The host cell according to claim 26, wherein the host cell is a eukaryotic cell.
28. A method of ing an antibody or a functional fragment of the antibody, characterized by comprising a step of culturing the host cell according to claim 26 or 27 and a step of collecting a desired antibody or a functional fragment of the antibody from a cultured product obtained in the culturing step.
29. An antibody or a functional nt of the antibody, characterized by being obtained by the production method according to claim 28.
30. The functional fragment of the antibody according to clain129, wherein.the functional fragment is selected.fronlthe group consisting of Fab, F(ab)2, Fab’ and Fv.
31. The dy or a functional fragment of the dy according to any one of claims 1 to 17, 29, and 30, wherein the modification of a glycan is regulated to enhance an antibody—dependent cellular cytotoxic ty.
32. A pharmaceutical composition characterized by comprising at least one of the antibodies or functional fragments of the antibodies according to any one of claims 1 to 17, and 29 to 31.
33. The ceutical composition according to claim 32, which is for treating a tumor.
34. A. pharmaceutical composition for treating' a tumor characterized by comprising at least one of the antibodies or functional fragments of the dies according to any one of claims 1 to 17 and 29 to 31 and at least one therapeutic agent for cancer.
35. The pharmaceutical composition according to claim 33 or 34, wherein the tumor is cancer.
36. The pharmaceutical composition according to claim 35, wherein the cancer is lung cancer, breast cancer, te cancer, pancreatic , colorectal cancer, a melanoma, liver cancer, ovarian.cancer, r cancer, stomach cancer, geal cancer, or kidney cancer.
37 . Use of an antibody or functional nt of the antibody according to claims 1 to 17 and 29 to 31 for the manufacture of a medicament for treating a tumor.
38 . Use of an antibody or functional fragment of the antibody according to claims 1 to 17 and 29 to 31 for the manufacture of aInedicament for use in.atnethod.of treating'a tumor wherein the method comprises administering the medicament and at least one therapeutic agent for cancer simultaneously, tely, or sequentially to an individual.
39. The use according to claim 37 or 38, wherein the tumor is‘cancer.
40. The use according to clain139, wherein.the cancer is lung cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, a melanoma, liver cancer, ovarian cancer, bladder cancer, h cancer, esophageal , or kidney cancer.
41. The antibody or a functional fragment of the antibody according to claim 1, substantially as herein described with reference to any one of the Examples and/or
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161478878P | 2011-04-25 | 2011-04-25 | |
| US61/478,878 | 2011-04-25 | ||
| JP2011-097645 | 2011-04-25 | ||
| JP2011097645 | 2011-04-25 | ||
| PCT/JP2012/060904 WO2012147713A1 (en) | 2011-04-25 | 2012-04-24 | Anti-b7-h3 antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NZ616809A NZ616809A (en) | 2015-08-28 |
| NZ616809B2 true NZ616809B2 (en) | 2015-12-01 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2012248470B2 (en) | Anti-B7-H3 antibody | |
| TWI751979B (en) | Anti-garp antibody | |
| JP2022515318A (en) | Antibodies and their uses | |
| EP2199390B1 (en) | Anti-epha2 antibody | |
| CN113227146B (en) | Claudin 18.2 binding moiety and uses thereof | |
| EP3020812B1 (en) | Novel anti-dr5 antibody | |
| JP2020504171A (en) | Anti-Tim-3 antibodies for combination with anti-PD-1 antibodies | |
| JP2020529863A (en) | Anti-ROR1 antibody and its preparation and usage | |
| WO2015076425A1 (en) | New monoclonal antibody | |
| WO2022237647A1 (en) | Binding molecule against dll3 and use thereof | |
| JP2015196665A (en) | Anti-lr11 monoclonal antibody having neutralizing activity and pharmaceutical comprising the same | |
| RU2809746C2 (en) | Humanized anti-vegf monoclonal antibody | |
| NZ616809B2 (en) | Anti-b7-h3 antibody | |
| EP4342914A1 (en) | Anti-bcam antibody or antigen-binding fragment thereof | |
| JP2015196666A (en) | Anti-lr11 monoclonal antibody having neutralizing activity and pharmaceutical comprising the same | |
| JP2015196667A (en) | Anti-lr11 monoclonal antibody having neutralizing activity and pharmaceutical comprising the same |