WO2012073985A1 - 細胞傷害誘導治療剤 - Google Patents
細胞傷害誘導治療剤 Download PDFInfo
- Publication number
- WO2012073985A1 WO2012073985A1 PCT/JP2011/077603 JP2011077603W WO2012073985A1 WO 2012073985 A1 WO2012073985 A1 WO 2012073985A1 JP 2011077603 W JP2011077603 W JP 2011077603W WO 2012073985 A1 WO2012073985 A1 WO 2012073985A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- region
- amino acid
- binding domain
- polypeptide
- fragment
- Prior art date
Links
- 230000003013 cytotoxicity Effects 0.000 title claims abstract description 27
- 231100000135 cytotoxicity Toxicity 0.000 title claims abstract description 27
- 239000003814 drug Substances 0.000 title claims description 32
- 229940124597 therapeutic agent Drugs 0.000 title claims description 29
- 230000001939 inductive effect Effects 0.000 title claims description 20
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 565
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 513
- 229920001184 polypeptide Polymers 0.000 claims abstract description 509
- 230000027455 binding Effects 0.000 claims abstract description 423
- 102000036639 antigens Human genes 0.000 claims abstract description 257
- 108091007433 antigens Proteins 0.000 claims abstract description 257
- 239000000427 antigen Substances 0.000 claims abstract description 255
- 125000000539 amino acid group Chemical group 0.000 claims description 239
- 210000004027 cell Anatomy 0.000 claims description 223
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims description 186
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims description 186
- 235000001014 amino acid Nutrition 0.000 claims description 157
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 126
- 238000000034 method Methods 0.000 claims description 112
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims description 110
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 109
- 108091008874 T cell receptors Proteins 0.000 claims description 106
- 150000001413 amino acids Chemical class 0.000 claims description 97
- 206010028980 Neoplasm Diseases 0.000 claims description 95
- 201000011510 cancer Diseases 0.000 claims description 77
- 108090000623 proteins and genes Proteins 0.000 claims description 75
- 230000001472 cytotoxic effect Effects 0.000 claims description 60
- 108010073807 IgG Receptors Proteins 0.000 claims description 34
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 33
- 102000009490 IgG Receptors Human genes 0.000 claims description 32
- 102000005962 receptors Human genes 0.000 claims description 32
- 108020003175 receptors Proteins 0.000 claims description 32
- 102000004169 proteins and genes Human genes 0.000 claims description 30
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 29
- 238000004519 manufacturing process Methods 0.000 claims description 29
- 235000018102 proteins Nutrition 0.000 claims description 29
- 239000004472 Lysine Substances 0.000 claims description 27
- 235000013922 glutamic acid Nutrition 0.000 claims description 23
- 239000004220 glutamic acid Substances 0.000 claims description 23
- 230000002829 reductive effect Effects 0.000 claims description 23
- 239000013598 vector Substances 0.000 claims description 23
- 108091033319 polynucleotide Proteins 0.000 claims description 22
- 239000002157 polynucleotide Substances 0.000 claims description 22
- 102000040430 polynucleotide Human genes 0.000 claims description 22
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 19
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 17
- 235000003704 aspartic acid Nutrition 0.000 claims description 17
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 17
- 239000012228 culture supernatant Substances 0.000 claims description 14
- 239000004480 active ingredient Substances 0.000 claims description 13
- 235000004279 alanine Nutrition 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 12
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 10
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 9
- 239000004475 Arginine Substances 0.000 claims description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 8
- 238000011282 treatment Methods 0.000 claims description 8
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 7
- 201000007270 liver cancer Diseases 0.000 claims description 7
- 208000014018 liver neoplasm Diseases 0.000 claims description 7
- 201000005202 lung cancer Diseases 0.000 claims description 7
- 208000020816 lung neoplasm Diseases 0.000 claims description 7
- 235000018417 cysteine Nutrition 0.000 claims description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 4
- 210000004899 c-terminal region Anatomy 0.000 claims description 4
- 239000012830 cancer therapeutic Substances 0.000 claims description 4
- 230000006378 damage Effects 0.000 claims description 4
- 239000004474 valine Substances 0.000 claims description 4
- 208000027418 Wounds and injury Diseases 0.000 claims description 3
- 231100000433 cytotoxic Toxicity 0.000 claims description 3
- 208000014674 injury Diseases 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 2
- 230000000259 anti-tumor effect Effects 0.000 abstract description 14
- 210000004369 blood Anatomy 0.000 abstract description 9
- 239000008280 blood Substances 0.000 abstract description 9
- 102000010956 Glypican Human genes 0.000 description 255
- 108050001154 Glypican Proteins 0.000 description 255
- 108050007237 Glypican-3 Proteins 0.000 description 255
- 229940024606 amino acid Drugs 0.000 description 133
- 238000012360 testing method Methods 0.000 description 52
- 239000013604 expression vector Substances 0.000 description 37
- 210000004408 hybridoma Anatomy 0.000 description 33
- 239000002299 complementary DNA Substances 0.000 description 28
- 230000000694 effects Effects 0.000 description 25
- 239000000243 solution Substances 0.000 description 25
- 108020004414 DNA Proteins 0.000 description 24
- 230000000875 corresponding effect Effects 0.000 description 24
- 230000014509 gene expression Effects 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 101100034502 Chlamydomonas reinhardtii ERY2 gene Proteins 0.000 description 17
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 16
- 239000012634 fragment Substances 0.000 description 16
- 230000001235 sensitizing effect Effects 0.000 description 16
- 238000006467 substitution reaction Methods 0.000 description 16
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 15
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 15
- 230000002209 hydrophobic effect Effects 0.000 description 15
- 239000011324 bead Substances 0.000 description 14
- 230000003053 immunization Effects 0.000 description 14
- 230000035772 mutation Effects 0.000 description 14
- 238000002649 immunization Methods 0.000 description 13
- 206010035226 Plasma cell myeloma Diseases 0.000 description 12
- 230000007910 cell fusion Effects 0.000 description 12
- 238000010586 diagram Methods 0.000 description 12
- 201000000050 myeloid neoplasm Diseases 0.000 description 12
- 239000008194 pharmaceutical composition Substances 0.000 description 12
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 12
- 108010076504 Protein Sorting Signals Proteins 0.000 description 11
- 239000002246 antineoplastic agent Substances 0.000 description 11
- 230000000295 complement effect Effects 0.000 description 11
- 238000012216 screening Methods 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 10
- 241000124008 Mammalia Species 0.000 description 10
- 230000008859 change Effects 0.000 description 10
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 241000282412 Homo Species 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 229960000419 catumaxomab Drugs 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000012636 effector Substances 0.000 description 9
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- 239000003446 ligand Substances 0.000 description 9
- 230000036961 partial effect Effects 0.000 description 9
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 9
- 108091008146 restriction endonucleases Proteins 0.000 description 9
- 108060003951 Immunoglobulin Proteins 0.000 description 8
- 108091005804 Peptidases Proteins 0.000 description 8
- 102000035195 Peptidases Human genes 0.000 description 8
- 230000010261 cell growth Effects 0.000 description 8
- 239000010432 diamond Substances 0.000 description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 8
- 230000004927 fusion Effects 0.000 description 8
- 210000002865 immune cell Anatomy 0.000 description 8
- 102000018358 immunoglobulin Human genes 0.000 description 8
- 239000002953 phosphate buffered saline Substances 0.000 description 8
- 241000283707 Capra Species 0.000 description 7
- 101000935587 Homo sapiens Flavin reductase (NADPH) Proteins 0.000 description 7
- 102000057297 Pepsin A Human genes 0.000 description 7
- 108090000284 Pepsin A Proteins 0.000 description 7
- 239000004365 Protease Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 230000002860 competitive effect Effects 0.000 description 7
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 7
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 7
- 229940111202 pepsin Drugs 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- -1 CD45RO Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 6
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000000824 cytostatic agent Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 238000005259 measurement Methods 0.000 description 6
- 230000036470 plasma concentration Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 102000005720 Glutathione transferase Human genes 0.000 description 5
- 108010070675 Glutathione transferase Proteins 0.000 description 5
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 5
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 5
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 description 5
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 102000001708 Protein Isoforms Human genes 0.000 description 5
- 108010029485 Protein Isoforms Proteins 0.000 description 5
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 5
- 230000001093 anti-cancer Effects 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 229960003008 blinatumomab Drugs 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000000796 flavoring agent Substances 0.000 description 5
- 235000013355 food flavoring agent Nutrition 0.000 description 5
- 108020001507 fusion proteins Proteins 0.000 description 5
- 102000037865 fusion proteins Human genes 0.000 description 5
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 102000009109 Fc receptors Human genes 0.000 description 4
- 108010087819 Fc receptors Proteins 0.000 description 4
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 4
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 description 4
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 4
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 4
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 239000012505 Superdex™ Substances 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 125000005842 heteroatom Chemical group 0.000 description 4
- 102000048373 human GPC3 Human genes 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 230000005917 in vivo anti-tumor Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000000314 lubricant Substances 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 238000007911 parenteral administration Methods 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 229940104230 thymidine Drugs 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 3
- 238000012492 Biacore method Methods 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 3
- 206010050685 Cytokine storm Diseases 0.000 description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 3
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 108090000270 Ficain Proteins 0.000 description 3
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 3
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 3
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 3
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 238000003016 alphascreen Methods 0.000 description 3
- 238000012440 amplified luminescent proximity homogeneous assay Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 206010052015 cytokine release syndrome Diseases 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000003113 dilution method Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 description 3
- 235000019836 ficin Nutrition 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000003966 growth inhibitor Substances 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000000375 suspending agent Substances 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- FXYPGCIGRDZWNR-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxopropyl]disulfanyl]propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSCCC(=O)ON1C(=O)CCC1=O FXYPGCIGRDZWNR-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- WQQBUTMELIQJNY-UHFFFAOYSA-N 1-[4-(2,5-dioxo-3-sulfopyrrolidin-1-yl)oxy-2,3-dihydroxy-4-oxobutanoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1CC(S(O)(=O)=O)C(=O)N1OC(=O)C(O)C(O)C(=O)ON1C(=O)CC(S(O)(=O)=O)C1=O WQQBUTMELIQJNY-UHFFFAOYSA-N 0.000 description 2
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 2
- 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
- QLHLYJHNOCILIT-UHFFFAOYSA-N 4-o-(2,5-dioxopyrrolidin-1-yl) 1-o-[2-[4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxobutanoyl]oxyethyl] butanedioate Chemical compound O=C1CCC(=O)N1OC(=O)CCC(=O)OCCOC(=O)CCC(=O)ON1C(=O)CCC1=O QLHLYJHNOCILIT-UHFFFAOYSA-N 0.000 description 2
- 102100027211 Albumin Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 101000953861 Bothrops erythromelas Zinc metalloproteinase-disintegrin-like berythractivase Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 102100036242 HLA class II histocompatibility antigen, DQ alpha 2 chain Human genes 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000930801 Homo sapiens HLA class II histocompatibility antigen, DQ alpha 2 chain Proteins 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 2
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 208000009625 Lesch-Nyhan syndrome Diseases 0.000 description 2
- 101710099301 Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 206010025538 Malignant ascites Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- WOUIMBGNEUWXQG-VKHMYHEASA-N Ser-Gly Chemical compound OC[C@H](N)C(=O)NCC(O)=O WOUIMBGNEUWXQG-VKHMYHEASA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102000013275 Somatomedins Human genes 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 2
- 108010087408 alpha-beta T-Cell Antigen Receptors Proteins 0.000 description 2
- 229960003896 aminopterin Drugs 0.000 description 2
- 238000012197 amplification kit Methods 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 210000000628 antibody-producing cell Anatomy 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- NXVYSVARUKNFNF-UHFFFAOYSA-N bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxybutanedioate Chemical compound O=C1CCC(=O)N1OC(=O)C(O)C(O)C(=O)ON1C(=O)CCC1=O NXVYSVARUKNFNF-UHFFFAOYSA-N 0.000 description 2
- VYLDEYYOISNGST-UHFFFAOYSA-N bissulfosuccinimidyl suberate Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)C(S(O)(=O)=O)CC1=O VYLDEYYOISNGST-UHFFFAOYSA-N 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 208000037887 cell injury Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- UHZZMRAGKVHANO-UHFFFAOYSA-M chlormequat chloride Chemical compound [Cl-].C[N+](C)(C)CCCl UHZZMRAGKVHANO-UHFFFAOYSA-M 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000007884 disintegrant Substances 0.000 description 2
- ZWIBGKZDAWNIFC-UHFFFAOYSA-N disuccinimidyl suberate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)CCC1=O ZWIBGKZDAWNIFC-UHFFFAOYSA-N 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 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 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 101150039713 gpc3 gene Proteins 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 210000001822 immobilized cell Anatomy 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000003094 microcapsule Substances 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 238000004091 panning Methods 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000006152 selective media Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 150000003457 sulfones Chemical class 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 239000011800 void material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- LAQPKDLYOBZWBT-NYLDSJSYSA-N (2s,4s,5r,6r)-5-acetamido-2-{[(2s,3r,4s,5s,6r)-2-{[(2r,3r,4r,5r)-5-acetamido-1,2-dihydroxy-6-oxo-4-{[(2s,3s,4r,5s,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}hexan-3-yl]oxy}-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy}-4-hydroxy-6-[(1r,2r)-1,2,3-trihydrox Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 LAQPKDLYOBZWBT-NYLDSJSYSA-N 0.000 description 1
- NDJNDUULNXNRQD-XKBRQERYSA-N 1-[(2r,4s,5s)-5-[bromo(hydroxy)methyl]-4-hydroxyoxolan-2-yl]pyrimidine-2,4-dione Chemical compound C1[C@H](O)[C@@H](C(Br)O)O[C@H]1N1C(=O)NC(=O)C=C1 NDJNDUULNXNRQD-XKBRQERYSA-N 0.000 description 1
- YYDMSFVTLYEPOH-UHFFFAOYSA-N 2,5-dioxo-1-propanoyloxypyrrolidine-3-sulfonic acid Chemical compound CCC(=O)ON1C(=O)CC(S(O)(=O)=O)C1=O YYDMSFVTLYEPOH-UHFFFAOYSA-N 0.000 description 1
- SYEKJCKNTHYWOJ-UHFFFAOYSA-N 2-(2,5-dioxopyrrolidin-1-yl)-2-sulfobutanedioic acid;ethane-1,2-diol Chemical compound OCCO.OC(=O)CC(S(O)(=O)=O)(C(O)=O)N1C(=O)CCC1=O.OC(=O)CC(S(O)(=O)=O)(C(O)=O)N1C(=O)CCC1=O SYEKJCKNTHYWOJ-UHFFFAOYSA-N 0.000 description 1
- DTFAJAKTSMLKAT-JDCCYXBGSA-N 2-deoxystreptamine Chemical compound N[C@H]1C[C@@H](N)[C@H](O)[C@@H](O)[C@@H]1O DTFAJAKTSMLKAT-JDCCYXBGSA-N 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- WYWHKKSPHMUBEB-UHFFFAOYSA-N 6-Mercaptoguanine Natural products N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 102100022749 Aminopeptidase N Human genes 0.000 description 1
- UIERETOOQGIECD-UHFFFAOYSA-N Angelic acid Natural products CC=C(C)C(O)=O UIERETOOQGIECD-UHFFFAOYSA-N 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 229940123205 CD28 agonist Drugs 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 102100036008 CD48 antigen Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 1
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102100025680 Complement decay-accelerating factor Human genes 0.000 description 1
- 102100030886 Complement receptor type 1 Human genes 0.000 description 1
- 102100032768 Complement receptor type 2 Human genes 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102100023471 E-selectin Human genes 0.000 description 1
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 1
- 108010021472 Fc gamma receptor IIB Proteins 0.000 description 1
- 108010021470 Fc gamma receptor IIC Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- LQEBEXMHBLQMDB-JGQUBWHWSA-N GDP-beta-L-fucose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C3=C(C(NC(N)=N3)=O)N=C2)O1 LQEBEXMHBLQMDB-JGQUBWHWSA-N 0.000 description 1
- 102000002702 GPI-Linked Proteins Human genes 0.000 description 1
- 108010043685 GPI-Linked Proteins Proteins 0.000 description 1
- 102100021260 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Human genes 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical class 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
- 108010024636 Glutathione Proteins 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 1
- 108010075704 HLA-A Antigens Proteins 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 1
- 101000757160 Homo sapiens Aminopeptidase N Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101000716130 Homo sapiens CD48 antigen Proteins 0.000 description 1
- 101000993364 Homo sapiens Ciliary neurotrophic factor Proteins 0.000 description 1
- 101000856022 Homo sapiens Complement decay-accelerating factor Proteins 0.000 description 1
- 101000727061 Homo sapiens Complement receptor type 1 Proteins 0.000 description 1
- 101000941929 Homo sapiens Complement receptor type 2 Proteins 0.000 description 1
- 101000622123 Homo sapiens E-selectin Proteins 0.000 description 1
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 1
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 1
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 description 1
- 101000932480 Homo sapiens Fms-related tyrosine kinase 3 ligand Proteins 0.000 description 1
- 101000894906 Homo sapiens Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Proteins 0.000 description 1
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000852815 Homo sapiens Insulin receptor Proteins 0.000 description 1
- 101001078158 Homo sapiens Integrin alpha-1 Proteins 0.000 description 1
- 101001078133 Homo sapiens Integrin alpha-2 Proteins 0.000 description 1
- 101000994378 Homo sapiens Integrin alpha-3 Proteins 0.000 description 1
- 101000994375 Homo sapiens Integrin alpha-4 Proteins 0.000 description 1
- 101000994369 Homo sapiens Integrin alpha-5 Proteins 0.000 description 1
- 101000994365 Homo sapiens Integrin alpha-6 Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101001046677 Homo sapiens Integrin alpha-V Proteins 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101000935040 Homo sapiens Integrin beta-2 Proteins 0.000 description 1
- 101001015004 Homo sapiens Integrin beta-3 Proteins 0.000 description 1
- 101000599852 Homo sapiens Intercellular adhesion molecule 1 Proteins 0.000 description 1
- 101000599858 Homo sapiens Intercellular adhesion molecule 2 Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101001055145 Homo sapiens Interleukin-2 receptor subunit beta Proteins 0.000 description 1
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 description 1
- 101000599056 Homo sapiens Interleukin-6 receptor subunit beta Proteins 0.000 description 1
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 1
- 101001129927 Homo sapiens Leptin receptor Proteins 0.000 description 1
- 101000942967 Homo sapiens Leukemia inhibitory factor Proteins 0.000 description 1
- 101000608935 Homo sapiens Leukosialin Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101000622137 Homo sapiens P-selectin Proteins 0.000 description 1
- 101001071312 Homo sapiens Platelet glycoprotein IX Proteins 0.000 description 1
- 101001070790 Homo sapiens Platelet glycoprotein Ib alpha chain Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 101000799461 Homo sapiens Thrombopoietin Proteins 0.000 description 1
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 description 1
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 101000622304 Homo sapiens Vascular cell adhesion protein 1 Proteins 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 208000018121 Hypoxanthine-guanine phosphoribosyltransferase deficiency Diseases 0.000 description 1
- 229940124753 IL-2 agonist Drugs 0.000 description 1
- 102100025323 Integrin alpha-1 Human genes 0.000 description 1
- 102100025305 Integrin alpha-2 Human genes 0.000 description 1
- 102100032819 Integrin alpha-3 Human genes 0.000 description 1
- 102100032818 Integrin alpha-4 Human genes 0.000 description 1
- 102100032817 Integrin alpha-5 Human genes 0.000 description 1
- 102100032816 Integrin alpha-6 Human genes 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 102100022337 Integrin alpha-V Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 102100025304 Integrin beta-1 Human genes 0.000 description 1
- 102100025390 Integrin beta-2 Human genes 0.000 description 1
- 102100032999 Integrin beta-3 Human genes 0.000 description 1
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 1
- 102100037872 Intercellular adhesion molecule 2 Human genes 0.000 description 1
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102100026879 Interleukin-2 receptor subunit beta Human genes 0.000 description 1
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 1
- 102100037795 Interleukin-6 receptor subunit beta Human genes 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 102100033467 L-selectin Human genes 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 1
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 1
- 102100039564 Leukosialin Human genes 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 102100029205 Low affinity immunoglobulin gamma Fc region receptor II-b Human genes 0.000 description 1
- 102100029206 Low affinity immunoglobulin gamma Fc region receptor II-c Human genes 0.000 description 1
- 102100029193 Low affinity immunoglobulin gamma Fc region receptor III-A Human genes 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 102100026964 M1-specific T cell receptor beta chain Human genes 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010031099 Mannose Receptor Proteins 0.000 description 1
- 108010087870 Mannose-Binding Lectin Proteins 0.000 description 1
- 102000009112 Mannose-Binding Lectin Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 101000993359 Mus musculus Ciliary neurotrophic factor Proteins 0.000 description 1
- 101000987583 Mus musculus Eosinophil peroxidase Proteins 0.000 description 1
- 101000920670 Mus musculus Erythropoietin Proteins 0.000 description 1
- 101000935589 Mus musculus Flavin reductase (NADPH) Proteins 0.000 description 1
- 101000932479 Mus musculus Fms-related tyrosine kinase 3 ligand Proteins 0.000 description 1
- 101000746384 Mus musculus Granulocyte colony-stimulating factor Proteins 0.000 description 1
- 101000852813 Mus musculus Insulin receptor Proteins 0.000 description 1
- 101001129925 Mus musculus Leptin receptor Proteins 0.000 description 1
- 101000942966 Mus musculus Leukemia inhibitory factor Proteins 0.000 description 1
- 101000836210 Mus musculus Somatotropin Proteins 0.000 description 1
- 101000799460 Mus musculus Thrombopoietin Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 102000003729 Neprilysin Human genes 0.000 description 1
- 108090000028 Neprilysin Proteins 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 102100023472 P-selectin Human genes 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 102100036851 Platelet glycoprotein IX Human genes 0.000 description 1
- 102100034173 Platelet glycoprotein Ib alpha chain Human genes 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- XZKQVQKUZMAADP-IMJSIDKUSA-N Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(O)=O XZKQVQKUZMAADP-IMJSIDKUSA-N 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N 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 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 102000036693 Thrombopoietin Human genes 0.000 description 1
- 108010041111 Thrombopoietin Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- VJHCJDRQFCCTHL-UHFFFAOYSA-N acetic acid 2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)=O.OCC(O)C(O)C(O)C(O)C=O VJHCJDRQFCCTHL-UHFFFAOYSA-N 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 102000006707 alpha-beta T-Cell Antigen Receptors Human genes 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 102000012740 beta Adrenergic Receptors Human genes 0.000 description 1
- 108010079452 beta Adrenergic Receptors Proteins 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 229950008138 carmellose Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000003163 cell fusion method Methods 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- CRVGKGJPQYZRPT-UHFFFAOYSA-N diethylamino acetate Chemical compound CCN(CC)OC(C)=O CRVGKGJPQYZRPT-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000012893 effector ligand Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- IYBKWXQWKPSYDT-UHFFFAOYSA-L ethylene glycol disuccinate bis(sulfo-N-succinimidyl) ester sodium salt Chemical compound [Na+].[Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCC(=O)OCCOC(=O)CCC(=O)ON1C(=O)C(S([O-])(=O)=O)CC1=O IYBKWXQWKPSYDT-UHFFFAOYSA-L 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 102000011778 gamma-delta T-Cell Antigen Receptors Human genes 0.000 description 1
- 108010062214 gamma-delta T-Cell Antigen Receptors Proteins 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000002523 gelfiltration 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
- 229960003180 glutathione Drugs 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 102000007318 human leptin receptor Human genes 0.000 description 1
- 230000005660 hydrophilic surface Effects 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000014726 immortalization of host cell Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 150000004667 medium chain fatty acids Chemical class 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 238000009520 phase I clinical trial Methods 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer 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
- 239000011148 porous material Substances 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- UIERETOOQGIECD-ONEGZZNKSA-N tiglic acid Chemical compound C\C=C(/C)C(O)=O UIERETOOQGIECD-ONEGZZNKSA-N 0.000 description 1
- MNRILEROXIRVNJ-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=NC=N[C]21 MNRILEROXIRVNJ-UHFFFAOYSA-N 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- 239000012929 tonicity agent Substances 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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
- C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
-
- 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
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- 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
- C07K16/18—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
- C07K16/303—Liver or Pancreas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/526—CH3 domain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
-
- 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
- C07K2317/71—Decreased effector function due to an Fc-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
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- the present invention provides a polypeptide aggregate, a method for producing the polypeptide aggregate, and a method for producing the polypeptide aggregate, by allowing T cells to be brought close to the target cancer cell and treating the cancer through cytotoxic activity of the T cell against the target cancer cell.
- the present invention relates to a cytotoxicity-inducing therapeutic agent comprising a peptide aggregate as an active ingredient.
- the present invention relates to a pharmaceutical composition for treating or preventing various cancers containing the cytotoxicity-inducing therapeutic agent as an active ingredient, or a therapeutic method using the pharmaceutical composition.
- Non-patent Document 1 A number of therapeutic antibodies exhibiting excellent antitumor effects have been developed as pharmaceuticals for cancer treatment. These therapeutic antibodies inhibit the signals necessary for cancer cell growth, induce cell death signals, or ADCC (Antibody Dependent Cell-mediated ity Cytotoxicity), CDC (Complement ; Dependent Cytotoxicity) It is known that an antitumor effect on cancer cells is exhibited by sexual cell injury (Non-patent Document 2). ADCC is the cytotoxicity that these effector cells exert against the target cancer cells bound by the antibody when the Fc region of the antibody binds to Fc receptors present on effector cells such as NK cells and macrophages. A complement complex binds to a complement binding site present in the structure of the antibody.
- ADCC Antibody Dependent Cell-mediated ity Cytotoxicity
- CDC Complement ; Dependent Cytotoxicity
- Complement components present in the complex form a pore on the cell membrane of the antibody-bound cell, which promotes the influx of water and ions into the cell and destroys the cell. is there.
- the existing therapeutic antibodies have excellent effects, the therapeutic results obtained by the administration of such antibodies are still unsatisfactory. Accordingly, development of therapeutic antibodies against cancers that exhibit even stronger cell killing activity is desired.
- T is an antibody that uses cytotoxicity, which recruits T cells as effector cells, as its mechanism for anti-tumor effects.
- Cell recruiting antibodies T cell recruiting antibodies, TR antibodies
- TR antibodies include antibodies to any of the constituent subunits of the T cell receptor (TCR) complex on T cells, particularly those that bind to the CD3 epsilon chain and antibodies that bind to the antigen on the target cancer cell. It is a bi-specific antibody.
- the TR antibody approaches the cancer cell by simultaneously binding to the CD3 ⁇ epsilon chain and the cancer antigen. As a result, it is considered that the antitumor effect on cancer cells is exhibited by the cytotoxic action of T cells.
- An antibody called trifunctional antibody is also known as one of TR antibodies (Non-patent Documents 6 and 7). This is a whole IgG-type bi-specific antibody in which a Fab that binds to a cancer antigen and a Fab that binds to a CD3 epsilon chain are contained in one arm.
- Catumaxomab a trifunctional antibody against EpCAM, has been shown to be effective in treating malignant ascites by administering intraperitoneally to malignant ascites patients with EpCAM expression-positive cancer cells.
- the EU has approved the use of catumaxomab for the above treatments.
- BiTE bispecific T-cell engagementr
- BiTE is a TR antibody having a molecular type in which scFv of an antibody against a cancer antigen and scFv of an antibody against a CD3 epsilon chain are linked via a short polypeptide linker.
- BiTE has been reported to have an antitumor action superior to various TR antibodies known so far (Non-Patent Documents 9 and 10). That is, BiTE exerts an anti-tumor effect at a significantly lower concentration and lower effector cell: cancer cell ratio (ET ratio) than other TR antibodies.
- ET ratio cancer cell ratio
- TR antibodies that recruit T cells as effector cells are usually used because catumaxomab is clinically approved and approved as a therapeutic drug, and multiple BiTEs including blinatumomab exert a strong antitumor effect. These results suggest that it has a very high potential as an anti-tumor agent compared to antibodies that have ADCC as its mechanism of action.
- BiTE unlike catumaxomab, does not have a binding site for the Fc ⁇ receptor, so that receptors expressed on T cells, NK cells, macrophages, etc. are not cross-linked independently of cancer antigens. Therefore, it has been shown that the induction of cancer antigen-independent cytokines observed when catumaxomab is administered does not occur.
- BiTE is a low-molecular weight modified antibody molecule lacking the Fc region, the blood half-life of BiTE administered to patients is significantly shorter than that of IgG-type antibodies normally used as therapeutic antibodies. There is a problem.
- Non-patent Documents 13 and 14 Non-patent Documents 13 and 14
- blinatumomab is administered by continuous intravenous administration using a minipump. Is being administered.
- Such administration is not only a very inconvenient administration method for patients, but also presents a risk of medical accidents due to equipment failure, and is not a desirable treatment method.
- the present invention has been made in view of the above circumstances, a polypeptide aggregate that allows T cells to be brought close to target cancer cells and treats cancer through cytotoxic activity against target cancer cells by T cells, It is an object of the present invention to provide a method for producing a polypeptide aggregate and a cytotoxicity-inducing therapeutic agent containing the polypeptide aggregate as an active ingredient. Moreover, it aims at providing the therapeutic method using the said pharmaceutical composition for treating or preventing various cancer containing the said cytotoxicity induction therapeutic agent as an active ingredient.
- the present inventors have maintained a strong antitumor activity of BiTE and an excellent safety property that does not induce cytokine storm and the like in a cancer antigen-independent manner, and a new polymorph with a long blood half-life. Peptide aggregates were found. Furthermore, it has been found that by substituting the antigen-binding domain in the polypeptide aggregate, the polypeptide aggregate causes cytotoxicity targeting various cells. Based on such findings, the present inventors have revealed that the polypeptide aggregate according to the present invention damages cancer cells. In addition, the present inventors have found that introduction of CH1 / CL interface association control and Knob into Hole (KiH) modification into a polypeptide aggregate causes cell damage more efficiently. Further, the present inventors have found that a cytotoxicity-inducing therapeutic agent comprising the polypeptide aggregate according to the present invention as an active ingredient treats or prevents various cancers.
- a cytotoxicity-inducing therapeutic agent comprising the polypeptide aggregate according to the present invention as an active ingredient treats or prevents various
- the present invention provides the following.
- the following domains (1) an antigen binding domain, (2) a domain containing an Fc region with reduced binding activity to an Fc ⁇ receptor, and (3) T cell receptor complex binding domain,
- a polypeptide assembly comprising: [2] The polypeptide complex according to [1], wherein the T cell receptor complex binding domain is a T cell receptor binding domain. [3] The polypeptide complex according to [1], wherein the T cell receptor complex binding domain is a CD3 binding domain. [4] The polypeptide aggregate according to any one of [1] to [3], wherein the antigen-binding domain is a bivalent antigen-binding domain.
- a monovalent scFv constitutes an Fc region via an scFv constituting a CD3 binding domain, and a heavy chain Fv fragment of a monovalent Fab constitutes an Fc region via a CH1 region.
- polypeptide aggregate according to any one of [1] to [3], wherein the antigen-binding domain is a bivalent scFv.
- a monovalent scFv is linked to one polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain, and the other monovalent scFv is linked to the other one polypeptide constituting the Fc region.
- each of the antigen-binding domain and the T cell receptor complex-binding domain is a monovalent Fab.
- a heavy chain Fv fragment of a monovalent Fab constituting an antigen binding domain is linked to one polypeptide constituting the Fc region via a CH1 region, and a light chain Fv fragment of the Fab is linked to a CL region.
- the heavy chain Fv fragment of Fab constituting the T cell receptor binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region,
- a heavy chain Fv fragment of a monovalent Fab constituting an antigen binding domain is linked to one polypeptide constituting the Fc region via a CH1 region, and a light chain Fv fragment of the Fab is linked to a CL region.
- the Fab light chain Fv fragment constituting the T cell receptor binding domain was linked to the other polypeptide constituting the Fc region via the CH1 region, and the Fab heavy chain Fv fragment was linked to the CL region, [22]
- a heavy chain Fv fragment of a monovalent Fab constituting the antigen binding domain is linked to one polypeptide constituting the Fc region via a CH1 region, and a light chain Fv fragment of the Fab is linked to a CL region.
- the heavy chain Fv fragment of Fab constituting the T cell receptor binding domain is linked to the other polypeptide constituting the Fc region via the CL region, and the light chain Fv fragment of the Fab is linked to the CH1 region, [22] The polypeptide aggregate according to [22]. [26] A heavy chain Fv fragment of a monovalent Fab constituting a T cell receptor binding domain is linked to one polypeptide constituting an Fc region via a CH1 region, and the light chain Fv fragment of the Fab is a CL region.
- the Fab light chain Fv fragment constituting the antigen-binding domain was linked to the other polypeptide constituting the Fc region via the CH1 region, and the Fab heavy chain Fv fragment was linked to the CL region, [22] The polypeptide aggregate according to [22].
- a heavy chain Fv fragment of a monovalent Fab constituting a T cell receptor binding domain is linked to one polypeptide constituting an Fc region via a CH1 region, and the light chain Fv fragment of the Fab is a CL region.
- the Fab heavy chain Fv fragment constituting the antigen binding domain is linked to the other polypeptide constituting the Fc region via the CL region, and the Fab light chain Fv fragment is linked to the CH1 region, [22] The polypeptide aggregate according to [22].
- a heavy chain Fv fragment of a monovalent Fab structure that binds to an antigen is linked to one polypeptide constituting the Fc region via a CH1 region, and the light chain Fv fragment of the Fab structure is CL An antigen-binding domain linked to a region
- a heavy chain Fv fragment of monovalent Fab structure that binds to the T cell receptor complex is linked to the other polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab structure is A T cell receptor complex binding domain linked to a CL region
- a polypeptide aggregate comprising: a heavy chain Fv fragment in an antigen binding domain and a light chain Fv fragment in an antigen binding domain or a heavy chain Fv fragment in a T cell receptor binding domain and a T cell receptor binding domain
- Amino acid residue of CH1 region linked to heavy chain Fv fragment in T cell receptor complex binding domain and amino acid residue of CL region linked to light chain Fv fragment in T cell receptor binding domain The polypeptide complex according to [29] or [31], wherein each has a charge different from each other.
- Both the amino acid residue of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residue of the CL region linked to the light chain Fv fragment in the antigen binding domain have different charges. [30] or the polypeptide aggregate according to [31].
- [36] The group consisting of one or two or more amino acid residues represented by the following (a) to (f), wherein the amino acid residues in the CH1 region and the CL region: (A) an amino acid residue at position 147 of the EU numbering in the CH1 region, and an amino acid residue at position 180 of the EU numbering in the CL region, (B) an amino acid residue in the CH1 region at position 147 EU numbering and an amino acid residue in the CL region at position 131 in EU numbering (c) an amino acid residue in the CH1 region EU numbering amino acid residue at position 147, and CL region amino acid residue at EU numbering position 164 (d) CH1 region amino acid residue at EU numbering position 147, and Amino acid residues in the CL region and EU numbering position 138 (e) Amino acid residues in the CH1 region and EU numbering position 147, and amino acid residues in the CL region and EU numbering The amino acid residue at position 123 (f) is selected from the amino acid residues in the CH1 region, EU number
- the group is any of the following groups (X) or (Y);
- the polypeptide complex according to [36] or [37] which is selected from the amino acid residues contained in.
- the amino acid residue having a heterogeneous charge is an amino acid residue in the CH1 region and the amino acid residue at position 175 of the EU is Lys, and the amino acid residue in the CL region is position No. 180 and 131 in the EU numbering
- the amino acid residues having different charges are amino acid residues in the CH1 region and EU numbering positions 147 and 175 are Glu, and the CL region is an amino acid residue and EU numbering position 180.
- the amino acid residue at position 213 in the EU numbering at position 213 in the CH1 region is Glu
- the amino acid residue at position 123 in the EU region at position number EU is Lys. 40].
- the Fc region constitutes the Fc region described in SEQ ID NO: 23, the Fc region described in SEQ ID NO: 24, the Fc region described in SEQ ID NO: 25, or the Fc region described in SEQ ID NO: 26
- the polypeptide aggregate according to any one of [1] to [42], which is an Fc region in which an amino acid is mutated.
- the polypeptide according to [43] wherein the amino acid sequence from position 118 to position 260 is the sequence shown in SEQ ID NO: 24, and the amino acid sequence from position 261 to position 447 is the Fc region shown in SEQ ID NO: 26. Association.
- the amino acid at position 349 specified according to EU numbering is cysteine
- the amino acid at position 366 is tryptophan
- the amino acid at position 356 specified according to EU numbering is cysteine
- the amino acid at position 366 is serine
- the amino acid at position 368 is alanine
- the amino acid at position 407 is mutated to valine
- the amino acid at position 356 specified according to EU numbering among the amino acid residues of one of the two polypeptides constituting the Fc region is lysine, and the amino acid residue of the other polypeptide is according to EU numbering
- the specified amino acid at position 439 is mutated to glutamic acid, and the amino acid at position 435 specified according to EU numbering among the amino acid residues of either polypeptide is mutated to arginine, [1 ]
- [53] The polypeptide aggregate according to [51] or [52], wherein the sequence GK present at the carboxy terminus of the two polypeptides constituting the Fc region is deleted.
- polypeptide aggregate according to [57] wherein a different epitope is present in the protein consisting of the amino acid sequence described in SEQ ID NO: 2.
- polypeptide aggregate according to [57] wherein a different epitope is present in the protein consisting of the amino acid sequence described in SEQ ID NO: 4.
- a vector comprising the polynucleotide according to [60].
- a method for producing a polypeptide aggregate comprising culturing the cell according to [62] and recovering the polypeptide aggregate from a culture supernatant.
- a cytotoxicity-inducing therapeutic agent comprising the polypeptide aggregate according to any one of [1] to [59] as an active ingredient.
- the therapeutic agent according to [64], wherein the cytotoxicity-inducing therapeutic agent is a cancer therapeutic agent.
- the therapeutic agent according to [65], wherein the cancer is liver cancer or lung cancer.
- a method for treating or preventing cancer comprising administering the polypeptide complex according to any one of [1] to [59] to a patient in need of treatment.
- the treatment or prevention method according to [67] wherein the cancer is liver cancer or lung cancer.
- the present invention also relates to a kit for use in the method of the present invention, comprising the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention.
- the present invention also relates to the use of the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention in the production of a cytotoxicity-inducing therapeutic agent.
- the present invention also relates to the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention for use in the method of the present invention.
- a novel polypeptide group that maintains the strong antitumor activity of BiTE and the excellent safety property that does not induce cytokine storm in a cancer antigen-independent manner and has a long blood half-life. Coalition was provided.
- the cytotoxicity-inducing therapeutic agent containing the polypeptide aggregate brings about cytotoxicity targeting various cells including cancer cells, Cancer can be treated or prevented.
- the patient not only is the safety high, but also a desirable treatment that is less burdensome and highly convenient can be performed.
- the black square ( ⁇ ) indicates the cytotoxic activity of GPC3 BiTE
- the black triangle ( ⁇ ) indicates the cytotoxic activity of GPC3 ERY8-2
- the white circle ( ⁇ ) indicates the cytotoxic activity of GPC3 ERY9-1
- the white square ( ⁇ ) indicates the cytotoxic activity of GPC3 ERY10-1.
- White squares ( ⁇ ) represent changes in tumor volume in the GPC3 ERY7 administration group.
- Black diamond ( ⁇ ) represents the change in tumor volume of the control group (PBS administration).
- FIG. 5 is a graph showing the in-vitro cytotoxic activity of GPC3 ERY18 L1, GPC3 ERY18L2, GPC3 ERY18L3, GPC3 ERY18L4, and GPC3 ERY18S1.
- the black triangle ( ⁇ ) is GPC3 ERY18 L1
- the black circle ( ⁇ ) is GPC3 ERY18 L2
- the black square ( ⁇ ) is GPC3 ERY18 L3
- the white square ( ⁇ ) is GPC3 ERY18 ⁇ L4
- the white diamond ( ⁇ ) is GPC3 ERY18 S1. Represents activity. It is a graph showing the comparison of the in-vitro cytotoxic activity of GPC3 ERY18 L3 and GPC3 ERY10-1.
- the black square ( ⁇ ) represents the cytotoxic activity of GPC3 ERY18 L3, and the white square ( ⁇ ) represents the cytotoxic activity of GPC3 ERY10-1.
- GPC3 ERY18 and GPC3 ERY19-3 are domain representations; domains represented by crossed lines are anti-cancer antigen (GPC3, EpCAM, EGFR) antibody heavy chain variable regions, domains represented by diagonal lines are anti-antibody Cancer antigen (GPC3, EpCAM, EGFR) antibody L chain variable region, domain indicated by dotted line is anti-CD3 antibody H chain variable region, domain indicated by black is anti-CD3 antibody L chain variable region, white Domain is an antibody constant region, a cross indicates a silent Fc mutation, and an asterisk indicates a mutation that associates a hetero Fc.
- FIG. 3 is a schematic diagram.
- GM1 or GM2 which are polypeptide aggregates described in the Example of this-application specification, and GM0.
- Polypeptide aggregates with CH1 / CL interface association control and Knob intoHole (KiH) modification introduced are designated as A, and polypeptide aggregates with neither CH1 / CL interface association control nor KiH introduced are designated as B.
- the domain represented by the cross line is represented by the anti-cancer antigen (GPC3, EpCAM) antibody heavy chain variable region, and the domain represented by the diagonal line is represented by the anti-cancer antigen (GPC3, EpCAM) antibody light chain variable region, dotted line Domain is the anti-CD3 antibody heavy chain variable region, the domain shown in black is the anti-CD3 antibody light chain variable region, the domain shown in white is the antibody constant region, the cross is the silent Fc mutation, and the asterisk is heterozygous Mutations that associate Fc and hollow circles represent mutations into which CH1 / CL interface association control is introduced.
- 3 is a graph showing a comparison of cytotoxic activities of GM1, GM2, and GM0.
- the black triangle ( ⁇ ) represents the cytotoxic activity of GM1
- the white square ( ⁇ ) represents the cytotoxic activity of GM2
- the white circle ( ⁇ ) represents the cytotoxic activity of GM0. It is a graph showing the cytotoxic activity of EGFR ERY17-2.
- the black triangle ( ⁇ ) represents the cytotoxic activity of EGFR®ERY17-2.
- an antibody refers to an immunoglobulin that is naturally occurring or produced by partial or complete synthesis.
- the antibody can be isolated from natural resources such as plasma and serum in which it naturally exists, or from the culture supernatant of hybridoma cells producing the antibody, or partially or completely by using techniques such as genetic recombination Can be synthesized.
- Preferred examples of antibodies include immunoglobulin isotypes and subclasses of those isotypes.
- human immunoglobulins nine classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM are known. Of these isotypes, the antibody of the present invention may include IgG1, IgG2, IgG3, and IgG4.
- a method for producing an antibody having a desired binding activity is known to those skilled in the art.
- a method for producing an antibody (anti-GPC3 antibody) that binds to GPC3 (Int J Cancer. (2003) 103 (4), 455-65) belonging to the GPI-anchored receptor family is exemplified below.
- An antibody that binds to an antigen other than GPC3 can also be appropriately produced according to the following examples.
- the anti-GPC3 antibody can be obtained as a polyclonal or monoclonal antibody using known means.
- a monoclonal antibody derived from a mammal can be preferably prepared.
- Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques.
- Monoclonal antibody-producing hybridomas can be prepared, for example, as follows by using known techniques. That is, a mammal is immunized according to a normal immunization method using GPC3 protein as a sensitizing antigen. The resulting immune cells are fused with known parental cells by conventional cell fusion methods. Next, hybridomas that produce anti-GPC3 antibodies can be selected by screening monoclonal antibody-producing cells by conventional screening methods.
- the production of a monoclonal antibody is performed as follows, for example.
- the GPC3 protein represented by 2) can be obtained. That is, a suitable host cell is transformed by inserting a gene sequence encoding GPC3 into a known expression vector.
- the desired human GPC3 protein is purified from the host cell or culture supernatant by a known method.
- GPC3 In order to obtain soluble GPC3 from the culture supernatant, for example, among the GPC3 polypeptide sequence represented by SEQ ID NO: 2, the GPI anchor sequence used for anchoring GPC3 on the cell membrane is used. A protein lacking 564-580 amino acids constituting the corresponding hydrophobic region is expressed instead of the GPC3 protein represented by SEQ ID NO: 2. Purified natural GPC3 protein can also be used as a sensitizing antigen as well.
- the purified GPC3 protein can be used as a sensitizing antigen used for immunization against mammals.
- a partial peptide of GPC3 can also be used as a sensitizing antigen.
- the partial peptide can also be obtained by chemical synthesis from the amino acid sequence of human GPC3. It can also be obtained by incorporating a part of the GPC3 gene into an expression vector for expression.
- the region and size of GPC3 peptide used as a partial peptide are not particularly limited to a specific embodiment.
- a preferred region can be selected from any amino acid sequence corresponding to 564-580 amino acids in the amino acid sequence of SEQ ID NO: 2.
- the number of amino acids constituting the peptide to be sensitized antigen is preferably at least 5 or more, for example 6 or more, or 7 or more. More specifically, a peptide having 8 to 50, preferably 10 to 30 residues can be used as a sensitizing antigen.
- a fusion protein obtained by fusing a desired partial polypeptide or peptide of GPC3 protein with a different polypeptide can be used as a sensitizing antigen.
- an antibody Fc fragment or a peptide tag can be suitably used.
- a vector that expresses a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in-frame and inserting the fusion gene into the expression vector as described above. The method for producing the fusion protein is described in Molecular® Cloning® 2nd® ed.
- GPC3 used as a sensitizing antigen and an immunization method using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693 and the like.
- the mammal immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion.
- rodent animals such as mice, rats, hamsters, rabbits, monkeys and the like are preferably used.
- the above animals are immunized with a sensitizing antigen.
- immunization is performed by administering a sensitizing antigen intraperitoneally or subcutaneously to a mammal.
- a sensitized antigen diluted with PBS (Phosphate-Buffered Saline) or physiological saline at an appropriate dilution ratio is mixed with a normal adjuvant, for example, Freund's complete adjuvant, and emulsified, if desired.
- the sensitizing antigen is administered to the mammal several times every 4 to 21 days.
- an appropriate carrier can be used during immunization with the sensitizing antigen.
- a partial peptide having a low molecular weight when used as a sensitizing antigen, it may be desirable to immunize the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.
- a carrier protein such as albumin or keyhole limpet hemocyanin.
- a hybridoma that produces a desired antibody can be prepared as follows using DNA immunization.
- DNA immunization a sensitized antigen is expressed in vivo in the immunized animal to which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal.
- This is an immunization method in which immune stimulation is given.
- the following advantages are expected in DNA immunization. -Maintains the structure of membrane proteins like GPC3 and can be given immune stimulation-No need to purify immune antigens
- DNA expressing GPC3 protein is first administered to an immunized animal.
- the DNA encoding GPC3 can be synthesized by a known method such as PCR.
- the obtained DNA is inserted into an appropriate expression vector and administered to an immunized animal.
- the expression vector for example, a commercially available expression vector such as pcDNA3.1 can be suitably used.
- a method for administering a vector to a living body a generally used method can be used.
- DNA immunization is performed by introducing gold particles adsorbed with an expression vector into cells of an immunized animal individual using a gene gun.
- an antibody recognizing GPC3 can also be prepared using the method described in International Publication WO2003 / 104453.
- immune cells are collected from the mammal and subjected to cell fusion. Spleen cells can be used as preferred immune cells.
- Mammalian myeloma cells are used as the cells fused with the immune cells.
- the myeloma cell is preferably provided with an appropriate selection marker for screening.
- a selectable marker refers to a trait that can (or cannot) survive under certain culture conditions.
- Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency).
- HGPRT deficiency hypoxanthine-guanine-phosphoribosyltransferase deficiency
- TK deficiency thymidine kinase deficiency
- Cells having HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity).
- HGPRT-deficient or TK-deficient cells can be selected in a medium containing 6 thioguanine, 8 azaguanine (hereinafter abbreviated as 8AG), or 5 'bromodeoxyuridine, respectively.
- 8AG 8 azaguanine
- 5 'bromodeoxyuridine normal cells that incorporate these pyrimidine analogs into DNA die.
- cells deficient in these enzymes that cannot take up these pyrimidine analogs can survive in selective media.
- G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) by a neomycin resistance gene.
- gentamicin analogs gentamicin analogs
- myeloma cells suitable for cell fusion are known.
- Examples of such myeloma cells include P3 (P3x63Ag8.653) (J.JImmunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1- 7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 ( Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133) and the like can be suitably used.
- P3x63Ag8.653 J.JImmunol. (1979) 123 (4)
- cell fusion between the immune cells and myeloma cells is performed according to a known method such as the method of Köhler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, for example, the cell fusion can be performed in a normal nutrient culture medium in the presence of a cell fusion promoter.
- a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary such as dimethyl sulfoxide is optionally added to increase the fusion efficiency.
- the usage ratio of immune cells and myeloma cells can be set arbitrarily.
- the number of immune cells is preferably 1 to 10 times that of myeloma cells.
- the culture medium used for the cell fusion for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture are used. Serum replacement fluid such as fetal serum (FCS) can be suitably added.
- FCS fetal serum
- a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60%. It is added at a concentration of (w / v).
- a desired fused cell is formed by gently mixing the mixture.
- cell fusion agents and the like that are undesirable for the growth of hybridomas can be removed by repeating the operation of adding the appropriate culture solution listed above and removing the supernatant by centrifugation.
- the hybridoma thus obtained can be selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine).
- a HAT culture solution a culture solution containing hypoxanthine, aminopterin and thymidine.
- the culture using the HAT culture solution can be continued for a time sufficient for cells other than the desired hybridoma (non-fused cells) to die (usually, sufficient time is several days to several weeks).
- screening and single cloning of hybridomas producing the desired antibody are performed by the usual limiting dilution method.
- the hybridoma thus obtained can be selected by using a selective culture solution corresponding to the selection marker possessed by the myeloma used for cell fusion.
- a selective culture solution corresponding to the selection marker possessed by the myeloma used for cell fusion.
- cells having HGPRT or TK deficiency can be selected by culturing in a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have succeeded in cell fusion with normal cells can selectively proliferate in the HAT medium.
- the culture using the HAT culture solution is continued for a time sufficient for cells other than the desired hybridoma (non-fusion cells) to die.
- a desired hybridoma can be selected by culturing for several days to several weeks. Subsequently, screening and single cloning of hybridomas producing the desired antibody can be performed by conventional limiting
- Desired antibody screening and single cloning can be suitably performed by a screening method based on a known antigen-antibody reaction.
- a monoclonal antibody that binds to GPC3 can bind to GPC3 expressed on the cell surface.
- Such monoclonal antibodies can be screened, for example, by FACS (fluorescence-activated cell sorting).
- FACS fluorescence-activated cell sorting
- cells expressing GPC3 are prepared.
- Preferred cells for screening are mammalian cells in which GPC3 is forcibly expressed.
- the binding activity of the antibody to GPC3 on the cell surface can be selectively detected. That is, a hybridoma that produces a GPC3 monoclonal antibody can be obtained by selecting a hybridoma that produces an antibody that does not bind to a host cell but binds to a GPC3 forced expression cell.
- the binding activity of the antibody to the immobilized GPC3-expressing cells can be evaluated based on the principle of ELISA.
- GPC3-expressing cells are immobilized in the well of an ELISA plate.
- the culture supernatant of the hybridoma is brought into contact with the immobilized cells in the well, and an antibody that binds to the immobilized cells is detected.
- the monoclonal antibody is derived from a mouse
- the antibody bound to the cell can be detected by an anti-mouse immunoglobulin antibody.
- a hybridoma that produces a desired antibody having an ability to bind to an antigen selected by these screenings can be cloned by a limiting dilution method or the like.
- the hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution.
- the hybridoma can also be stored for a long time in liquid nitrogen.
- the hybridoma is cultured according to a usual method, and a desired monoclonal antibody can be obtained from the culture supernatant.
- a hybridoma can be administered to a mammal compatible therewith and allowed to proliferate, and a monoclonal antibody can be obtained from the ascites.
- the former method is suitable for obtaining a highly pure antibody.
- An antibody encoded by an antibody gene cloned from antibody-producing cells such as the hybridoma can also be suitably used.
- An antibody encoded by the gene is expressed by incorporating the cloned antibody gene into a suitable vector and introducing it into a host. Methods for isolation of antibody genes, introduction into vectors, and transformation of host cells are already established by, for example, Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767- 775). As described below, methods for producing recombinant antibodies are also known.
- cDNA encoding a variable region (V region) of an anti-GPC3 antibody is obtained from a hybridoma cell that produces the anti-GPC3 antibody.
- V region variable region
- RNA is extracted from the hybridoma.
- the following method can be used. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)
- Extracted mRNA can be purified using mRNA “Purification” Kit (manufactured by GE Healthcare Bioscience) or the like.
- kits for extracting total mRNA directly from cells such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience) are also commercially available.
- mRNA can be obtained from the hybridoma.
- CDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase.
- cDNA can be synthesized by AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation).
- the desired cDNA fragment is purified from the obtained PCR product and then ligated with vector DNA.
- a desired recombinant vector can be prepared from Escherichia coli that has formed the colony. Then, whether or not the recombinant vector has the target cDNA base sequence is confirmed by a known method such as the dideoxynucleotide chain termination method.
- cDNA is synthesized using RNA extracted from a hybridoma cell as a template to obtain a 5'-RACE cDNA library.
- a commercially available kit such as SMART® RACE® cDNA® amplification kit is appropriately used for the synthesis of the 5′-RACE® cDNA library.
- the antibody gene is amplified by the PCR method using the obtained 5'-RACE ⁇ ⁇ ⁇ ⁇ ⁇ cDNA library as a template.
- Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different nucleotide sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as IsoIStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics).
- primers capable of amplifying genes encoding ⁇ 1, ⁇ 2a, ⁇ 2b, ⁇ 3 as heavy chains and ⁇ and ⁇ chains as light chains are provided. Can be used.
- a primer that anneals to a portion corresponding to a constant region close to the variable region is generally used as the 3 ′ primer.
- the primer attached to the 5 ′ RACE cDNA library preparation kit is used as the 5 ′ primer.
- an immunoglobulin comprising a combination of a heavy chain and a light chain
- Desired antibodies can be screened using the reconstituted immunoglobulin binding activity to GPC3 as an index.
- the binding of the antibody to GPC3 is more preferably specific.
- Antibodies that bind to GPC3 can be screened, for example, as follows; (1) contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with a GPC3-expressing cell; (2) a step of detecting the binding between the GPC3-expressing cell and the antibody, and (3) a step of selecting an antibody that binds to the GPC3-expressing cell.
- a method for detecting the binding between an antibody and a GPC3-expressing cell is known. Specifically, the binding between the antibody and the GPC3-expressing cell can be detected by a technique such as FACS described above. In order to evaluate the binding activity of the antibody, a fixed specimen of GPC3-expressing cells can be appropriately used.
- a panning method using a phage vector is also preferably used as an antibody screening method using binding activity as an index.
- an antibody gene is obtained from a polyclonal antibody-expressing cell group as a library of heavy and light chain subclasses
- a screening method using a phage vector is advantageous.
- Genes encoding the variable regions of the heavy chain and the light chain can form a single chain Fv (scFv) by ligating with an appropriate linker sequence.
- scFv single chain Fv
- the phage encoding the antigen can be recovered to recover the DNA encoding scFv having the desired binding activity. By repeating this operation as necessary, scFv having a desired binding activity can be concentrated.
- the cDNA is digested with a restriction enzyme that recognizes restriction enzyme sites inserted at both ends of the cDNA.
- a preferred restriction enzyme recognizes and digests a base sequence that appears infrequently in the base sequence constituting the antibody gene.
- a restriction enzyme that gives a sticky end.
- An antibody expression vector can be obtained by inserting a cDNA encoding the V region of the anti-GPC3 antibody digested as described above into an appropriate expression vector.
- a chimeric antibody is obtained.
- the chimeric antibody means that the origin of the constant region and the variable region are different.
- a heterologous chimeric antibody such as mouse-human
- a human-human homologous chimeric antibody is also included in the chimeric antibody of the present invention.
- a chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector having a constant region in advance.
- a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately arranged on the 5 ′ side of an expression vector holding a DNA encoding a desired antibody constant region (C region).
- a chimeric antibody expression vector is constructed by fusing both digested with the same combination of restriction enzymes in-frame.
- the antibody gene is incorporated into an expression vector so that it is expressed under the control of the expression control region.
- An expression control region for expressing an antibody includes, for example, an enhancer and a promoter.
- An appropriate signal sequence can also be added to the amino terminus so that the expressed antibody is secreted extracellularly.
- a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 72) is used as the signal sequence, but other suitable signal sequences are added.
- the expressed polypeptide can be cleaved at the carboxyl terminal portion of the sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide.
- an appropriate host cell is transformed with this expression vector, whereby a recombinant cell expressing a DNA encoding an anti-GPC3 antibody can be obtained.
- DNAs encoding antibody heavy chains (H chains) and light chains (L chains) are each incorporated into separate expression vectors.
- An antibody molecule having an H chain and an L chain can be expressed by co-transfecting the same host cell with a vector in which an H chain and an L chain are incorporated.
- host cells can be transformed by incorporating DNAs encoding H and L chains into a single expression vector (see International Publication WO 94/11523).
- host cells and expression vectors for producing antibodies by introducing an isolated antibody gene into a suitable host are known. Any of these expression systems can be applied to isolate the antigen-binding domain and CD3-binding domain of the present invention.
- animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, the following cells can be exemplified as animal cells.
- Mammalian cells CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc.
- Amphibian cells Xenopus oocytes, etc.
- Insect cells sf9, sf21, Tn5, etc.
- Nicotiana such as Nicotiana tabacum
- Callus cultured cells can be used as appropriate for transformation of plant cells.
- -Yeast Saccharomyces genus such as Saccharomyces serevisiae, Pichia genus such as methanol-utilizing yeast (Pichia pastoris)-Filamentous fungi: Aspergillus genus such as Aspergillus niger
- antibody gene expression systems using prokaryotic cells are also known.
- bacterial cells such as E. coli and Bacillus subtilis can be appropriately used.
- An expression vector containing the target antibody gene is introduced into these cells by transformation. By culturing the transformed cells in vitro, a desired antibody can be obtained from the culture of the transformed cells.
- transgenic animals can also be used for the production of recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding a desired antibody has been introduced.
- an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein that is uniquely produced in milk.
- a protein secreted in milk for example, goat ⁇ casein can be used.
- the DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat.
- the desired antibody can be obtained as a fusion protein with milk protein from milk produced by a transgenic goat (or its progeny) born from a goat that has received the embryo.
- hormones can be administered to transgenic goats to increase the amount of milk containing the desired antibody produced from the transgenic goat (Bio / Technology (1994), 12 (7), 699-702). .
- polypeptide assembly described in the present specification When the polypeptide assembly described in the present specification is administered to humans, it is a genetically modified form that has been artificially modified for the purpose of reducing the heterologous antigenicity to humans as an antigen-binding domain in the assembly.
- An antigen-binding domain derived from an antibody can be appropriately employed.
- the recombinant antibody includes, for example, a humanized antibody. These modified antibodies are appropriately produced using known methods.
- variable region of an antibody used to generate an antigen binding domain in a polypeptide assembly described herein typically comprises three complementarity determining regions (four) sandwiched between four framework regions (FR). complementarity-determining (region); (CDR).
- CDRs are regions that substantially determine the binding specificity of an antibody.
- the amino acid sequence of CDR is rich in diversity.
- the amino acid sequences constituting FR often show high identity even among antibodies having different binding specificities. Therefore, it is generally said that the binding specificity of a certain antibody can be transplanted to another antibody by CDR grafting.
- Humanized antibodies are also referred to as reshaped human antibodies.
- non-human animals for example, humanized antibodies obtained by grafting mouse antibody CDRs to human antibodies are known.
- General genetic recombination techniques for obtaining humanized antibodies are also known.
- Overlap-Extension-PCR is known as a method for transplanting mouse antibody CDRs into human FRs.
- PCR extension the base sequence which codes CDR of the mouse antibody which should be transplanted is added to the primer for synthesize
- a human FR comprising an amino acid sequence having high identity with the FR amino acid sequence adjacent to the mouse CDR to be transplanted.
- the base sequences to be linked are designed to be connected to each other in frame.
- Human FRs are synthesized individually by each primer.
- a product in which DNA encoding mouse CDR is added to each FR is obtained.
- the base sequences encoding mouse CDRs of each product are designed to overlap each other.
- the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed with each other to perform a complementary chain synthesis reaction. By this reaction, human FRs are linked via the mouse CDR sequence.
- a human-type antibody expression vector can be prepared by inserting the DNA obtained as described above and a DNA encoding the human antibody C region into an expression vector so as to be fused in frame. After introducing the integration vector into a host to establish a recombinant cell, the recombinant cell is cultured, and a DNA encoding the humanized antibody is expressed, whereby the humanized antibody is cultured in the cultured cell. (See European Patent Publication EP 239400, International Publication WO1996 / 002576).
- the CDR forms a favorable antigen-binding site when linked via CDR.
- a human antibody FR can be suitably selected.
- FR amino acid residues can be substituted so that the CDR of the reshaped human antibody forms an appropriate antigen-binding site.
- amino acid sequence mutations can be introduced into FRs by applying the PCR method used for transplantation of mouse CDRs into human FRs.
- partial nucleotide sequence mutations can be introduced into primers that anneal to the FR.
- a nucleotide sequence mutation is introduced into the FR synthesized by such a primer.
- a mutant FR sequence having a desired property can be selected by measuring and evaluating the antigen-binding activity of a mutant antibody substituted with an amino acid by the above method (Sato, K.et al., Cancer Res, 1993, 53, 851-856).
- transgenic animals having all repertoires of human antibody genes are used as immunized animals, and DNA immunization is performed. Desired human antibodies can be obtained.
- the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method.
- Phages expressing scFv that bind to the antigen can be selected.
- the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined.
- the V region sequence is fused in-frame with the sequence of the desired human antibody C region, and then inserted into an appropriate expression vector, whereby an expression vector can be prepared.
- the human antibody is obtained by introducing the expression vector into a suitable expression cell as described above and expressing the gene encoding the human antibody.
- These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).
- Antigen-binding domain refers to a part of an antibody that comprises a region that specifically binds and is complementary to a part or all of an antigen. When the molecular weight of the antigen is large, the antibody can only bind to a specific part of the antigen. The specific part is called an epitope.
- the antigen binding domain may be provided from one or more antibody variable domains. Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
- antigen binding domains examples include “scFv (single chain Fv)”, “single chain antibody”, “Fv”, “scFv2 (single chain Fv 2)”, “Fab” or “F ( ab ′) 2 ”and the like are preferable.
- the antigen-binding domain in the polypeptide aggregate of the present invention can bind to the same epitope.
- the same epitope can be present in a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
- the antigen binding domains in the polypeptide assembly of the present invention can bind to different epitopes.
- different epitopes can exist in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.
- the specific specific, specifically one molecule of molecules that bind refers to a state that does not show any significant binding to molecules other than molecules of the other party to one or more binding thereof. Moreover, it is used also when an antigen binding domain is specific with respect to a specific epitope among several epitopes contained in a certain antigen. In addition, when an epitope to which an antigen binding domain binds is contained in a plurality of different antigens, a polypeptide assembly having the antigen binding domain can bind to various antigens including the epitope.
- the antigen is not particularly limited and may be any antigen except CD3.
- the antigen preferably include a receptor, a cancer antigen, an MHC antigen, and a differentiation antigen.
- receptors include, for example, hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase type receptor family, TNF receptor family, G protein coupled receptor family, GPI Examples include receptors belonging to the receptor family such as anchor type receptor family, tyrosine phosphatase type receptor family, adhesion factor family, hormone receptor family, and the like.
- Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptors (Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277- 285), human or mouse granulocyte colony stimulating factor (G-CSF) receptor (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341-350), human or mouse thrombopoietin (TPO) receptor (Proc Natl Acad Sci U S A. (1992) 89 (12), 5640-5644, EMBO J.
- EPO erythropoietin
- human or mouse leptin receptor human or mouse growth hormone (GH) receptor, human or mouse Interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human or mouse ciliary neurotrophic factor (CNTF) receptor A body etc. are illustrated suitably.
- GH growth hormone
- IL Interleukin
- IGF insulin-like growth factor
- LIF human or mouse leukemia inhibitory factor
- CNTF ciliary neurotrophic factor
- Cancer antigens are antigens that are expressed as cells become malignant and are also called tumor-specific antigens.
- abnormal sugar chains appearing on the cell surface and protein molecules when cells become cancerous are also cancer antigens and are also called cancer sugar chain antigens.
- cancer antigens include, for example, GPC3 (Int J Cancer. (2003) 103 (4) that belongs to the GPI-anchored receptor family as the above receptor but is expressed in several cancers including liver cancer. , 455-65), EpCAM expressed in multiple cancers including lung cancer (Proc1989Natl Acad Sci U S A. ⁇ ⁇ (1989) 86 (1), 27-31) (its polynucleotide sequence is RefSeq accession number NM_002354.
- polypeptide sequence is described in RefSeq accession number NP_002345.2 (SEQ ID NO: 4), respectively), EGFR, CA19-9, CA15-3, serial SSEA-1 (SLX ) Etc. are mentioned suitably.
- MHC antigens are mainly classified into MHC class I antigen and MHC class II antigen, which includes HLA-A, -B, -C, -E, -F, -G, -H.
- MHC class II antigens include HLA-DR, -DQ, and -DP.
- an epitope which refers to an antigenic determinant present in an epitope antigen, refers to a site on an antigen to which an antigen binding domain in a polypeptide assembly disclosed herein binds.
- an epitope can be defined by its structure.
- the epitope can also be defined by the binding activity to the antigen in the polypeptide aggregate that recognizes the epitope.
- the antigen is a peptide or polypeptide
- the epitope can be specified by the amino acid residues constituting the epitope.
- the epitope is a sugar chain
- the epitope can be specified by a specific sugar chain structure.
- a linear epitope is an epitope including an epitope whose primary amino acid sequence is recognized.
- Linear epitopes typically include at least 3, and most commonly at least 5, for example about 8 to about 10, 6 to 20 amino acids in a unique sequence.
- a conformational epitope is, in contrast to a linear epitope, an epitope in which the primary sequence of the amino acid containing the epitope is not a single defining component of the recognized epitope (eg, the primary sequence of amino acids does not necessarily define the epitope).
- a conformational epitope may include an increased number of amino acids relative to a linear epitope.
- the antibody recognizes the three-dimensional structure of the peptide or protein. For example, when a protein molecule is folded to form a three-dimensional structure, certain amino acid and / or polypeptide backbones that form a conformational epitope are juxtaposed to allow the antibody to recognize the epitope.
- Methods for determining the conformation of an epitope include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope® Mapping® Protocols in Methods Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).
- the method for confirming binding to an epitope by a test polypeptide aggregate containing an antigen binding domain for GPC3 is exemplified below. Confirmation of binding to an epitope by a test polypeptide aggregate containing an antigen binding domain for an antigen other than GPC3
- the method can also be appropriately implemented according to the following examples.
- a test polypeptide aggregate containing an antigen-binding domain for GPC3 recognizes a linear epitope present in a GPC3 molecule.
- a linear peptide consisting of an amino acid sequence constituting the extracellular domain of GPC3 is synthesized.
- the peptide can be chemically synthesized.
- it can be obtained by genetic engineering techniques using a region encoding an amino acid sequence corresponding to the extracellular domain in GPC3 cDNA.
- the binding activity between the linear peptide consisting of the amino acid sequence constituting the extracellular domain and the test polypeptide aggregate containing the antigen-binding domain for GPC3 is evaluated.
- the binding activity of the polypeptide aggregate to the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen.
- the binding activity to the linear peptide can be revealed based on the level of inhibition by the linear peptide in binding of the polypeptide aggregate to GPC3-expressing cells. These tests can reveal the binding activity of the polypeptide aggregates to linear peptides.
- test polypeptide aggregate containing an antigen-binding domain for GPC3 recognizes a three-dimensional epitope.
- cells expressing GPC3 are prepared.
- substantially not binding means 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% or less of the binding activity to human GPC3-expressing cells.
- a test polypeptide aggregate containing an antigen-binding domain to GPC3 to GPC3-expressing cells for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359 -420). That is, it can be evaluated by the principle of ELISA or FACS (fluorescence-activated cell sorting) using GPC3-expressing cells as antigens.
- the binding activity of the test polypeptide aggregate containing the antigen-binding domain for GPC3 to GPC3-expressing cells is quantitatively evaluated by comparing the signal level generated by the enzymatic reaction. That is, a test polypeptide aggregate is added to an ELISA plate on which GPC3-expressing cells are immobilized, and the test polypeptide aggregate bound to the cell is detected using an enzyme-labeled antibody that recognizes the test polypeptide aggregate.
- FACS the binding activity of test polypeptide aggregates to GPC3-expressing cells can be compared by preparing a dilution series of test polypeptide aggregates and determining the antibody binding titer (titer) for GPC3-expressing cells. .
- the binding of the test polypeptide aggregate to the antigen expressed on the cell surface suspended in a buffer or the like can be detected by a flow cytometer.
- a flow cytometer For example, the following devices are known as flow cytometers.
- EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)
- the following method may be mentioned as an example of a suitable method for measuring the binding activity of a test polypeptide aggregate containing an antigen binding domain for GPC3 to an antigen.
- a test polypeptide aggregate containing an antigen binding domain for GPC3 to an antigen.
- a suitable buffer As appropriate, the aggregate is adjusted to a desired concentration and used. For example, it can be used at any concentration between 10 ⁇ g / ml and 10 ng / ml.
- fluorescence intensity and cell number are measured by FACSCalibur (BD).
- the amount of antibody bound to the cells is reflected in the fluorescence intensity obtained by analysis using CELL QUEST Software (BD), that is, the value of Geometric Mean. That is, by obtaining the value of Geometric Mean, the binding activity of the test polypeptide aggregate represented by the binding amount of the test polypeptide aggregate can be measured.
- BD CELL QUEST Software
- the test polypeptide is prepared after the GPC3 protein coated on the wells of the microtiter plate is pre-incubated in the presence or absence of candidate competitive polypeptide aggregates. Aggregates are added.
- the amount of test polypeptide aggregate bound to the GPC3 protein in the well is indirectly correlated with the binding ability of candidate competitive polypeptide aggregates that compete for binding to the same epitope. That is, the greater the affinity of the competitive polypeptide aggregate for the same epitope, the lower the binding activity of the test polypeptide aggregate to the well coated with the GPC3 protein.
- the amount of the test polypeptide aggregate bound to the well via the GPC3 protein can be easily measured by labeling the polypeptide aggregate in advance.
- biotin-labeled polypeptide aggregates are measured by using an avidin peroxidase conjugate and an appropriate substrate.
- a cross-blocking assay using an enzyme label such as peroxidase is particularly referred to as a competitive ELISA assay.
- Polypeptide aggregates can be labeled with other labeling substances that can be detected or measured. Specifically, radiolabels or fluorescent labels are known.
- the competing polypeptide assembly at least binds the test polypeptide assembly comprising the antigen binding domain to GPC3. If it can block 20%, preferably at least 20-50%, more preferably at least 50%, the test polypeptide assembly binds to or substantially binds to the same epitope as the competitor polypeptide assembly. Polypeptide aggregates that compete for binding.
- test polypeptide assembly and the control polypeptide assembly share the epitope. It can be evaluated by comparing the binding activities of both polypeptide aggregates to peptides in which amino acid mutations are introduced into the constituent peptides.
- binding activity for example, it can be measured by comparing the binding activity of a test polypeptide aggregate and a control polypeptide aggregate to a linear peptide into which a mutation has been introduced in the ELISA format described above.
- a method for adsorbing a mutant peptide on a column as a fusion peptide with GST, for example, is known.
- the identified epitope is a steric epitope
- cells that express GPC3 and cells that express GPC3 in which a mutation is introduced into the epitope are prepared.
- a test polypeptide aggregate and a control polypeptide aggregate are added to a cell suspension in which these cells are suspended in an appropriate buffer such as PBS.
- a FITC-labeled antibody capable of recognizing the test polypeptide aggregate and the control polypeptide aggregate is added to the cell suspension appropriately washed with the buffer.
- the fluorescence intensity and the number of cells stained with the labeled antibody are measured by FACSCalibur (BD).
- the concentration of the test polypeptide aggregate and the control polypeptide aggregate is adjusted to a desired concentration by appropriately diluting with a suitable buffer and used. For example, it is used at any concentration between 10 ⁇ g / ml and 10 ng / ml.
- the amount of the labeled antibody bound to the cells is reflected in the fluorescence intensity obtained by analysis using CELL
- “substantially does not bind to mutant GPC3-expressing cells” can be determined by the following method. First, the test polypeptide aggregate and the control polypeptide aggregate bound to the cells expressing the mutant GPC3 are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. By calculating this comparison value ( ⁇ Geo-Mean) based on the following formula from the value of Geometric Mean in the presence and absence of polypeptide aggregates, increase in fluorescence intensity due to binding of polypeptide aggregates The percentage can be determined.
- ⁇ Geo-Mean Geo-Mean (in the presence of polypeptide aggregate) / Geo-Mean (in the absence of polypeptide aggregate)
- Analytical Geometric Mean comparison value (mutated GPC3 molecule ⁇ Geo-Mean value) that reflects the amount of binding of test polypeptide aggregates to mutant GPC3-expressing cells obtained by analysis reflects the amount of binding of test polypeptide aggregates to GPC3-expressing cells Compare with the ⁇ Geo-Mean comparison value.
- concentrations of the test polypeptide aggregates used in determining the ⁇ Geo-Mean comparison value for the mutant GPC3-expressing cell and the GPC3-expressing cell are adjusted to the same or substantially the same concentration.
- a polypeptide aggregate that has been confirmed to recognize an epitope in GPC3 in advance is used as a control polypeptide aggregate.
- ⁇ Geo-Mean comparison value for the mutant GPC3-expressing cell of the test polypeptide aggregate is at least 80%, preferably 50%, more preferably 30% of the ⁇ Geo-Mean comparison value for the GPC3-expressing cell of the test polypeptide aggregate, Particularly preferably, if it is less than 15%, it means “does not substantially bind to mutant GPC3-expressing cells”.
- the calculation formula for obtaining the Geo-Mean value (Geometric Mean) is described in CELL QUEST Software User's Guide (BD biosciences).
- the epitope of the test polypeptide assembly and the control polypeptide assembly can be assessed to be the same if it is such that it can be substantially equated by comparing the comparison values.
- Fv (variable fragment) refers to a pair of a light chain variable region (VL) of an antibody and a heavy chain variable region (VH) of an antibody. Is the smallest unit of an antigen-binding domain derived from an antibody.
- VL light chain variable region
- VH heavy chain variable region
- Fv for example, the following polypeptide aggregates;
- the monovalent scFv is composed of one polypeptide constituting the Fc region via the heavy chain Fv fragment constituting the CD3 binding domain, and the other monovalent scFv is the light chain constituting the CD3 binding domain.
- the bivalent antigen-binding domain linked to the other polypeptide constituting the Fc region via the Fv fragment is a bivalent scFv (1) a bivalent antigen-binding domain, (2) IgG1, IgG2a, A domain containing an Fc region that does not have an Fc ⁇ receptor binding activity among amino acids constituting the Fc region of IgG3 or IgG4, and (3) at least a monovalent CD3 binding domain, A pair of Fvs in which a light chain Fv fragment and a heavy chain Fv fragment are associated with each other in a form having a binding to CD3 as an antigen to form a CD3 binding domain is also preferably included.
- single chain antibodies includes variable regions derived from both heavy and light chains within a single polypeptide chain. Means an antibody fragment lacking the constant region.
- single chain antibodies further comprise a polypeptide linker between the VH and VL domains that enables the formation of the desired structure that would allow antigen binding.
- Single chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, 113, Rosenburg, and Moore, Springer-Verlag, New York, 269-315 (1994). Similarly, see International Patent Application Publication No. WO1988 / 001649 and US Pat. Nos. 4,946,778 and 5,260,203.
- single chain antibodies can also be bispecific and / or humanized.
- ScFv is an antigen binding domain in which VH and VL constituting Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. (1988) 85 (16), 5879-5883). VH and VL can be held in close proximity by the peptide linker.
- sc (Fv) 2 is a single-chain antibody in which four variable regions of two VLs and two VHs are connected by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1- 2), 177-189).
- the two VHs and VLs can be derived from different monoclonal antibodies.
- the bispecific recognition (bispecific sc (Fv) 2) that recognizes two kinds of epitopes existing in the same antigen as disclosed in JournalJof Immunology (1994) 152 (11), 5368-5374 is also preferable.
- sc (Fv) 2 can be produced by methods known to those skilled in the art. For example, it can be prepared by linking scFv with a linker such as a peptide linker.
- the antigen-binding domain constituting sc (Fv) 2 in the present specification includes two VHs and two VLs, VH, VL, VH, VL ([[ VH] linker [VL] linker [VH] linker [VL]) are listed in this order, but the order of two VH and two VL is not particularly limited to the above configuration, They may be arranged in any order. For example, the following configuration can be given.
- sc (Fv) 2 The molecular form of sc (Fv) 2 is also described in detail in WO2006 / 132352, and those skilled in the art will be able to produce a polypeptide assembly disclosed herein based on these descriptions. It is possible to produce a desired sc (Fv) 2 as appropriate.
- the polypeptide aggregate of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent.
- a sugar chain addition sequence can be inserted, and a sugar chain can be suitably added for the purpose of obtaining a desired effect.
- a peptide linker is preferable.
- the length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. However, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably Is 20 amino acids or less), particularly preferably 15 amino acids.
- sc (Fv) 2 includes three peptide linkers, peptide linkers having the same length may be used, or peptide linkers having different lengths may be used.
- Synthetic chemical linkers are commonly used for cross-linking peptides such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) Suberate (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene Glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2- (succinimideoxycarbonyloxy ) Ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy
- linkers When linking four antibody variable regions, usually three linkers are required, but all may use the same linker or different linkers.
- Fab, F (ab ') 2, or Fab' “Fab” is composed of one light chain and the CH1 and variable regions of one heavy chain.
- the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
- F (ab ') 2" and “Fab'” are produced by treating immunoglobulin (monoclonal antibody) with proteolytic enzymes such as pepsin or papain, and between the two H chains in the hinge region.
- H chain fragment consisting of the chain and VH (H chain variable region) and CH ⁇ 1 ( ⁇ 1 region in the H chain constant region) is linked by a disulfide bond in the C-terminal region.
- Fab ' Each of these two homologous antibody fragments is called Fab '.
- F (ab ') 2' ' includes two light chains and two constant regions containing constant regions of the CH1 domain and part of the CH2 domain such that an interchain disulfide bond is formed between the two heavy chains. Contains heavy chains.
- F (ab ′) 2 constituting the polypeptide aggregate disclosed in the present specification is obtained by partially digesting a full-length monoclonal antibody or the like having a desired antigen-binding domain with a proteolytic enzyme such as pepsin. It can be suitably obtained by adsorbing on a protein A column and removing it.
- Such a proteolytic enzyme is not particularly limited as long as it can digest a full-length antibody so as to produce F (ab ′) 2 restrictively by appropriately setting the reaction conditions of the enzyme such as pH.
- pepsin and ficin can be exemplified.
- the Fc region constituting the polypeptide aggregate disclosed in the present specification is obtained by partially digesting an antibody such as a monoclonal antibody with a protease such as pepsin, and then adsorbing the fragment to a protein A column or a protein G column. Then, it can be suitably obtained by elution with an appropriate elution buffer or the like.
- a proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH, and examples thereof include pepsin and ficin.
- the polypeptide aggregate described in the present specification includes an Fc region having a reduced binding activity to the Fc ⁇ receptor among amino acids constituting the Fc region of IgG1, IgG2, IgG3, or IgG4.
- the antibody isotype is determined by the structure of the constant region.
- the constant regions of each isotype of IgG1, IgG2, IgG3, and IgG4 are called C ⁇ 1, C ⁇ 2, C ⁇ 3, and C ⁇ 4, respectively.
- the amino acid sequences of the polypeptides constituting the Fc regions of human C ⁇ 1, C ⁇ 2, C ⁇ 3, and C ⁇ 4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26.
- the relationship between the amino acid residues constituting each amino acid sequence and kabat's EU numbering (also referred to herein as EU INDEX) is shown in FIG.
- the Fc region includes two light chains and two heavy chains that include a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains. This is the area excluding (ab ') 2.
- the Fc region constituting the polypeptide aggregate disclosed in the present specification is the fraction adsorbed on the protein A column after partial digestion of IgG1, IgG2, IgG3, IgG4 monoclonal antibody, etc. with a protease such as pepsin. It can be suitably obtained by re-eluting the minutes.
- Such a proteolytic enzyme is not particularly limited as long as it can digest a full-length antibody so as to produce F (ab ′) 2 restrictively by appropriately setting the reaction conditions of the enzyme such as pH.
- pepsin and ficin can be exemplified.
- Fc ⁇ receptor Fc ⁇ receptor is a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and means virtually any member of the protein family encoded by the Fc ⁇ receptor gene. To do.
- this family includes Fc ⁇ RI (CD64) including isoforms Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc; isoforms Fc ⁇ RIIa (including allotypes H131 and R131), Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2) and Fc ⁇ RIIc Fc ⁇ RII (CD32); and isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2), and any undiscovered human Fc ⁇ Rs or Fc ⁇ R isoforms Although allotype is also included, it is not limited to these.
- Fc ⁇ R may be derived from any organism, including but not limited to humans, mice, rats, rabbits and monkeys.
- Mouse Fc ⁇ Rs include Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16) and Fc ⁇ RIII-2 (CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes. It is not limited to.
- Preferable examples of such Fc ⁇ receptors include human Fc ⁇ I (CD64), Fc ⁇ IIA (CD32), Fc ⁇ IIB (CD32), Fc ⁇ IIIA (CD16) and / or Fc ⁇ IIIB (CD16).
- the polynucleotide sequence and amino acid sequence of Fc ⁇ I are shown in SEQ ID NOs: 13 (NM_000566.3) and 14 (NP_000557.1), respectively.
- the polynucleotide sequence and amino acid sequence of Fc ⁇ IIA are shown in SEQ ID NOs: 15 (BC020823.1) and 16 ( AAH20823.1), the polynucleotide sequence and amino acid sequence of Fc ⁇ IIB are SEQ ID NO: 17 (BC146678.1) and 18 (AAI46679.1), respectively, and the polynucleotide sequence and amino acid sequence of Fc ⁇ IIIA are SEQ ID NO: 19 (BC033678, respectively).
- Fc ⁇ IIIB polynucleotide sequence and amino acid sequence of Fc ⁇ IIIB are described in SEQ ID NOs: 21 (BC128562.1) and 22 (AAI28563.1), respectively, (RefSeq in parentheses) Registration number).
- Fc ⁇ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies, in addition to the FACS and ELISA formats described above, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and surface It can be confirmed by the BIACORE method using the plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
- SPR plasmon resonance
- Fc ligand or “effector ligand” means a molecule, preferably a polypeptide, derived from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. Binding of the Fc ligand to Fc preferably induces one or more effector functions.
- Fc ligands include Fc receptor, Fc ⁇ R, Fc ⁇ R, Fc ⁇ R, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus protein G and viral Fc ⁇ R. However, it is not limited to these.
- Fc ligands also include Fc receptor homologues (FcRH), a family of Fc receptors homologous to Fc ⁇ R (Davisaviet al., (2002) Immunological Reviews 190, 123-136). Fc ligands can also include undiscovered molecules that bind to Fc.
- FcRH Fc receptor homologues
- Fc ⁇ R Fc receptor homologues
- Fc ligands can also include undiscovered molecules that bind to Fc.
- the Fc ⁇ receptor binding activity Fc region has reduced Fc ⁇ I, Fc ⁇ IIA, Fc ⁇ IIB, Fc ⁇ IIIA and / or Fc ⁇ IIIB Fc ⁇ receptor binding activity, in addition to the FACS and ELISA formats described above, It can be confirmed by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) or BIACORE method using surface plasmon resonance (SPR) phenomenon (Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010 ).
- ALPHA screen is implemented based on the following principle by ALPHA technology using two beads of donor and acceptor.
- a molecule bound to the donor bead interacts biologically with the molecule bound to the acceptor bead, and a luminescent signal is detected only when the two beads are in close proximity.
- a photosensitizer in the donor bead excited by the laser converts ambient oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor bead, and when it reaches the adjacent acceptor bead, it causes a chemiluminescence reaction in the bead, and finally light is emitted.
- the chemiluminescence reaction does not occur because the singlet oxygen produced by the donor bead does not reach the acceptor bead.
- biotin-labeled polypeptide aggregates are bound to donor beads, and Fc ⁇ receptors tagged with glutathione S-transferase (GST) are bound to acceptor beads.
- GST glutathione S-transferase
- the polypeptide aggregate having a wild type Fc region interacts with the Fc ⁇ receptor to produce a signal of 520-620 nm.
- Polypeptide aggregates with untagged mutant Fc regions compete with the interaction between polypeptide aggregates with wild-type Fc regions and Fc ⁇ receptors. Relative binding affinity can be determined by quantifying the decrease in fluorescence that results from competition.
- biotinylate polypeptide aggregates such as antibodies using Sulfo-NHS-biotin and the like.
- a method of tagging the Fc ⁇ receptor with GST it is expressed in a cell or the like holding a fusion gene in which a polynucleotide encoding the Fc ⁇ receptor and a polynucleotide encoding GST are fused in-frame.
- a method of purification using a glutathione column can be appropriately employed.
- the obtained signal is suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).
- the Biacore system takes the shift amount, that is, the mass change at the sensor chip surface on the vertical axis, and displays the time change of mass as measurement data (sensorgram).
- Kinetics association rate constant (ka) and dissociation rate constant (kd) are obtained from the sensorgram curve, and affinity (KD) is obtained from the ratio of the constants.
- an inhibition measurement method is also preferably used. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.
- the binding activity to the Fc ⁇ receptor is reduced, for example, based on the analysis method described above, compared to the competitive activity of the polypeptide aggregate as a control
- Competitive activity is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7 % Or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
- polypeptide aggregate used as a control a polypeptide aggregate having an Fc region of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be used as appropriate.
- the structure of the Fc region is as follows: SEQ ID NO: 23 (A added to the N terminus of RefSeq registration number AAC82527.1), 24 (A added to the N terminus of RefSeq registration number AAB59393.1), 25 (RefSeq registration number CAA27268.1) A (addition of A to the N terminus), 26 (RefSeq registration number AAB59394.1, A addition to the N ending).
- polypeptide aggregate having a variant of the Fc region of an antibody of a specific isotype When a polypeptide aggregate having a variant of the Fc region of an antibody of a specific isotype is used as a test substance, the polypeptide aggregate having the Fc region of the antibody of the specific isotype is used as a control. Thus, the effect of the mutation of the mutant on the binding activity to the Fc ⁇ receptor is verified. As described above, a polypeptide aggregate having a variant of the Fc region that has been verified to have reduced binding activity to the Fc ⁇ receptor is appropriately prepared.
- mutants include deletion of 231A-238S, an amino acid identified according to EU numbering (WO 2009/011941), C226S, C229S, P238S, (C220S) (J.Rheumatol (2007) 34, 11), C226S, C229S (Hum.Antibod.Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192) It is known.
- any one of the following amino acids specified according to EU numbering positions 220, 226, 229, 231, 232, 233, 234 , 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 297, 298, 299
- Preferred examples include polypeptide aggregates having Fc regions in which the position, position 300, position 325, position 327, position 328, position 329, position 330, position 331, and position 332 are substituted.
- the isotype of the antibody originating from the Fc region is not particularly limited, and an Fc region originating from an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as appropriate, but an Fc region originating from an IgG1 antibody is preferably used. Is done.
- amino acids constituting the Fc region of IgG1 antibody one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid positioned before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (A) L234F, L235E, P331S, (B) C226S, C229S, P238S, (C) C226S, C229S, (D) C226S, C229S, E233P, L234V, L235A Polypeptide aggregates having an Fc region to which an amino acid sequence has been applied, or an Fc region from which the amino acid sequence at positions 231 to 238 has been deleted can also be used as appropriate.
- EU numbering position of amino acid residue specified according to EU numbering, one-letter amino acid positioned before the number
- the symbol is the amino acid residue before substitution
- the one-letter amino acid symbol located after the number represents the
- amino acids constituting the Fc region of IgG2 antibody one of the following substitutions specified according to EU numbering (number of amino acid residue specified according to EU numbering, one-letter amino acid located before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (E) H268Q, V309L, A330S, P331S (F) V234A (G) G237A (H) V234A, G237A (I) A235E, G237A (J) V234A, A235E, G237A Polypeptide aggregates having an Fc region that has been subjected to can also be used as appropriate.
- amino acids constituting the Fc region of IgG3 antibody one of the following substitutions specified according to EU numbering (number of amino acid residue specified according to EU numbering, one-letter amino acid located before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (K) F241A (L) D265A (M) V264A Polypeptide aggregates having an Fc region that has been subjected to can also be used as appropriate.
- amino acids constituting the Fc region of IgG4 antibody one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid located before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (N) L235A, G237A, E318A (O) L235E (P) F234A, L235A Polypeptide aggregates having an Fc region that has been subjected to can also be used as appropriate.
- any one of the following amino acids specified according to EU numbering 233, 234, 235, 236, 237, 327, 327, 330
- Polypeptide aggregates that have an Fc region in which the EU numbering is substituted with the corresponding amino acid in the corresponding IgG2 or IgG4 at position 331.
- any one or more of the following amino acids specified according to EU numbering; positions 234, 235 and 297 are substituted with other amino acids
- Preferred examples include polypeptide aggregates having the Fc region.
- the type of amino acid present after substitution is not particularly limited, but a polypeptide aggregate having an Fc region in which any one or more amino acids at positions 234, 235, and 297 are substituted with alanine is particularly preferable.
- any one of the following amino acids specified according to EU numbering; polypeptide aggregate having Fc region substituted at position 265 with another amino acid are preferable.
- the type of amino acid present after the substitution is not particularly limited, but a polypeptide aggregate having an Fc region in which the amino acid at position 265 is substituted with alanine is particularly preferable.
- bispecific antibodies are antibodies that have two different specificities.
- An IgG-type bispecific antibody can be secreted by hybrid hybridoma (quadroma) produced by fusing two hybridomas producing IgG antibody (Milstein C et al. Nature (1983) 305, 537-540). .
- the IgG type bispecific antibody is secreted by introducing a total of four types of L chain and H chain genes constituting the two types of IgG of interest into a cell and co-expressing them.
- L chain and H chain genes constituting the two types of IgG of interest
- the secreted amount of the target combination is theoretically significantly reduced, a large culture scale is required, and the production cost further increases.
- IgG having a heterogeneous combination with the H chain can be preferentially secreted.
- the amino acid side chain present in the CH3 region of one H chain is replaced with a larger side chain (knob (meaning “protrusion”)), and the amino acid side present in the CH3 region of the other H chain
- knock meaning “protrusion”
- hole meaning “void”
- the L chain since the diversity of the L chain variable region is lower than that of the H chain variable region, it is expected that a common L chain capable of giving binding ability to both H chains can be obtained.
- the common L chain and both H chain genes By introducing the common L chain and both H chain genes into cells, the expression of IgG enables efficient expression of bispecific IgG (Nature Biotechnology IV (1998) 16, 77 677-681).
- a common L chain corresponding to any different H chain and exhibiting high binding ability is selected.
- a selection method has also been proposed (WO2004 / 065611).
- bispecific antibodies by using a method for controlling the association of polypeptides or heterologous multimers composed of polypeptides for the association of two polypeptides constituting the Fc region.
- a method for controlling the association of polypeptides or heterologous multimers composed of polypeptides for the association of two polypeptides constituting the Fc region are known. That is, by altering the amino acid residues that form the interface in the two polypeptides constituting the Fc region, the association of the polypeptides constituting the Fc region having the same sequence is inhibited, and two Fc regions having different sequences
- a method of controlling the formation of a polypeptide aggregate that constitutes can be employed for the production of bispecific antibodies (WO2006 / 106905).
- two polypeptides constituting the Fc region originating from the above-mentioned bispecific antibody can be used as appropriate. More specifically, in the two polypeptides constituting the Fc region, the amino acid at position 349 specified according to EU numbering in the amino acid sequence of one of the polypeptides is cysteine, and the amino acid at position 366 is tryptophan.
- the amino acid sequence of the other polypeptide identified by EU numbering is cysteine, amino acid at position 366 is serine, amino acid at position 368 is alanine, and amino acid at position 407 is valine. Two polypeptides are preferably used.
- the domain containing the Fc region according to the present invention includes two polypeptides constituting the Fc region, the amino acid sequence of one of the polypeptides at position 409 specified according to EU numbering.
- Two polypeptides are preferably used, wherein the amino acid is aspartic acid, and the amino acid at position 399 specified according to EU numbering in the amino acid sequence of the other polypeptide is lysine.
- the amino acid at position 409 may be glutamic acid instead of aspartic acid
- the amino acid at position 399 may be arginine instead of lysine.
- 360th aspartic acid or 392rd aspartic acid can also be suitably added.
- the domain containing the Fc region according to the present invention includes two polypeptides constituting the Fc region, the amino acid at position 370 identified according to EU numbering in the amino acid sequence of one of the polypeptides. Is preferably glutamic acid, and two polypeptides characterized in that the amino acid at position 357 specified according to EU numbering in the amino acid sequence of the other polypeptide is lysine.
- the domain containing the Fc region according to the present invention includes two polypeptides constituting the Fc region, and the amino acid sequence of one of the polypeptides at position 439 specified according to EU numbering.
- Two polypeptides are preferably used, wherein the amino acid is glutamic acid and the amino acid at position 356 specified according to EU numbering in the amino acid sequence of the other polypeptide is lysine.
- the two polypeptides constituting the Fc region wherein the amino acid at position 409 specified according to EU numbering in the amino acid sequence of one of the polypeptides is aspartic acid, the amino acid at position 370 is glutamic acid, and the other polypeptide
- Two polypeptides (aspartic acid in this embodiment instead of glutamic acid at position 370), characterized in that the amino acid at position 399 identified according to EU numbering is lysine and the amino acid at position 357 is lysine.
- polypeptides constituting the Fc region wherein the amino acid at position 409 specified according to EU numbering in the amino acid sequence of one polypeptide is aspartic acid, the amino acid at position 439 is glutamic acid, and the other polypeptide Amino acid sequence according to EU numbering, the two amino acids characterized in that the amino acid at position 399 is lysine and the amino acid at position 356 is lysine (in this embodiment, instead of glutamic acid at position 439, Aspartic acid, aspartic acid at position 392 or aspartic acid at position 439),
- the two polypeptides constituting the Fc region wherein the amino acid at position 370 specified according to EU numbering in one of the polypeptides is glutamic acid, the amino acid at position 439 is glutamic acid, and the other polypeptide
- the domain containing the Fc region according to the present invention includes two polypeptides constituting the Fc region, and the amino acid sequence of one of the polypeptides is identified at position 356 according to EU numbering.
- the second amino acid is preferably lysine, and the other polypeptide is characterized in that the amino acid at position 435 specified according to EU numbering in the amino acid sequence of the other polypeptide is arginine and the amino acid at position 439 is glutamic acid. It is done.
- the antigen-binding domain and / or the CD3 binding domain according to the present invention is obtained. It becomes possible to arrange in a desired combination.
- the Cc-terminal heterogeneity of the Fc region is improved in addition to the above characteristics as an Fc region with reduced Fc ⁇ receptor binding activity.
- the Fc region can be used as appropriate. More specifically, glycine at position 446 and lysine at position 447, which are specified according to EU numbering, are deleted from the amino acid sequences of two polypeptides constituting the Fc region originating from IgG1, IgG2, IgG3 or IgG4. Fc region is provided.
- T cell receptor complex binding domain refers to a region that specifically binds to and is complementary to part or all of a T cell receptor complex. A portion of a T cell receptor complex antibody comprising.
- the T cell receptor complex may be the T cell receptor itself or an adapter molecule that constitutes the T cell receptor complex together with the T cell receptor.
- a suitable adapter is CD3.
- T cell receptor binding domain refers to a T cell receptor comprising a region that specifically binds to and is complementary to part or all of a T cell receptor. The part of the body antibody.
- the T cell receptor may be a variable region or a constant region, but a preferred epitope to which a CD3 binding domain binds is an epitope present in the constant region.
- constant region sequences include T cell receptor ⁇ chain (SEQ ID NO: 67) of RefSeq accession number CAA26636.1, T cell receptor ⁇ chain of RefSeq accession number C25777 (SEQ ID NO: 68), and RefSeq accession number A26659.
- T cell receptor ⁇ 1 chain (SEQ ID NO: 69), RefSeq accession number AAB63312.1 T cell receptor ⁇ 2 chain (SEQ ID NO: 70), RefSeq accession number AAA61033.1 T cell receptor ⁇ chain (SEQ ID NO: 71).
- CD3 binding domain refers to the portion of a CD3 antibody that comprises a region that specifically binds and is complementary to part or all of CD3.
- the CD3 binding domain can be provided from one or more antibody variable domains.
- the CD3 binding domain comprises a light chain variable region (VL) of a CD3 antibody and a heavy chain variable region (VH) of a CD3 antibody.
- VL light chain variable region
- VH heavy chain variable region
- Examples of such CD3 binding domains include “scFv (single chain Fv)”, “single chain antibody”, “Fv”, “scFv2 (single chain Fv 2)”, “Fab” or “F ( ab ′) 2 ”and the like are preferable.
- CD3 binding can bind to any epitope as long as it is an epitope present in the ⁇ chain, ⁇ chain, or ⁇ chain sequence constituting human CD3.
- CD3 binding preferably comprises a light chain variable region (VL) of a CD3 antibody that binds to an epitope present in the extracellular region of the ⁇ chain of a human CD3 complex and a heavy chain variable region (VH) of a CD3 antibody. Domains are preferably used.
- VL light chain variable region
- VH heavy chain variable region
- CD3 binding domains include the OKT3 antibody (Proc. Natl. Acad. Sci.
- a CD3 binding domain comprising a region (VH) is preferably used.
- a CD3 binding domain originating from a CD3 antibody having a desired property obtained by immunizing a desired animal with a ⁇ chain, ⁇ chain, or ⁇ chain constituting human CD3 by the above method can be used as appropriate.
- an appropriately humanized antibody or a human antibody as described above is appropriately used.
- the structure of the ⁇ chain, ⁇ chain, or ⁇ chain constituting CD3 is such that the polynucleotide sequence is SEQ ID NO: 27 (NM_000073.2), 29 (NM_000732.4) and 31 (NM_000733.3).
- the sequences are described in SEQ ID NOs: 28 (NP_000064.1), 30 (NP_000723.1) and 32 (NP_000724.1) (the parentheses indicate RefSeq registration numbers).
- polypeptide aggregates are as described above.
- an antigen binding domain (2) a domain containing an Fc region with reduced binding activity to an Fc ⁇ receptor, and (3) T cell receptor complex binding domain,
- the structure is not limited, as long as it contains any one.
- the T cell receptor complex binding domain is preferably a T cell receptor binding domain or a CD3 binding domain.
- Each of the above domains can be directly linked by a peptide bond.
- F (ab ') 2 when (1) F (ab ') 2 is used as an antigen-binding domain, and (2) these Fc regions are used as a domain containing an Fc region with reduced Fc ⁇ receptor binding activity (1)
- the antigen-binding domain described in (1) and the domain containing the Fc region described in (2) are linked by a peptide bond, the linked polypeptides form an antibody structure.
- the antibody in addition to purifying from the above-mentioned hybridoma culture solution, the antibody is obtained from a culture solution of a desired host cell in which a polynucleotide encoding the polypeptide constituting the antibody is stably retained. It can also be purified.
- the CD3 binding domain can be bound to the C-terminus of the constant region of the antibody structure via a peptide bond.
- the CD3 binding domain may be bound via a peptide bond to the N-terminus of the heavy chain variable region or light chain variable region of the antibody structure.
- the CD3 binding domain can be linked via a peptide bond to the C-terminus of the light chain constant region of the antibody structure.
- a CD3 binding domain having a desired structure can be adopted as the CD3 binding domain to be bound, but Fv, preferably scFv, is suitably used.
- the valence of the CD3 binding domain that binds to the antibody structure is not limited.
- bivalent CD3-binding domain In order to bind a bivalent CD3-binding domain to the antibody structure, a monovalent CD3-binding domain is bound to each C-terminus of the two Fc regions constituting the constant region of the antibody structure via a peptide bond. obtain.
- bivalent scFv that is, sc (Fv) 2 binds to the C-terminus of one of the two Fc regions via a peptide bond.
- a bivalent scFv is only present at the C-terminus of one Fc region of the two Fc regions constituting the constant region of the antibody structure.
- a polypeptide aggregate to which sc (Fv) 2 binds is efficiently obtained.
- a monovalent scFv can be bound to the C terminus of one Fc region of two Fc regions via a peptide bond.
- a monovalent scFv only at the C-terminus of one Fc region of the two Fc regions constituting the constant region of the antibody structure. Is efficiently obtained.
- the heavy chain Fv fragment constituting the CD3 binding domain is one constant region constituting the Fc region.
- Polypeptide aggregates linked to the C-terminus (CH3 domain) of the other constant region of the Fc region are also used as appropriate. Is done.
- a linker such as Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 7) is appropriately inserted when linking the heavy chain Fv fragment or the light chain Fv fragment to the C-terminus (CH3 domain) of the constant region.
- the number of linker repeats is not limited and is selected from 1 to 10, preferably 2 to 8, and further 2 to 6 numbers, ie 1, 2, 3, 4, 5, 6, 7, 8, 9 or A linker such as Gly / Gly / Gly / Gly / Ser (SEQ ID NO: 7) consisting of 10 repeats can be inserted as appropriate.
- the heavy chain Fv fragment constituting the CD3 binding domain is linked to the C-terminus (CH3 domain) of one constant region constituting the Fc region, and the light chain Fv fragment constituting the CD3 binding domain constitutes the Fc region.
- the heavy chain Fv fragment is used to enhance the association between the heavy chain Fv fragment and the light chain Fv fragment. Modification of amino acid residues so as to form a disulfide bond between the Fc fragment and the light chain Fv fragment can also be performed as appropriate.
- the heavy chain Fv fragment constituting the CD3 binding domain is linked to the C terminus (CH3 domain) of one constant region constituting the Fc region, and the light chain Fv fragment constituting the CD3 binding domain is linked to the Fc region.
- the heavy chain Fv fragment is combined with the light chain Fv fragment in order to enhance the association between the heavy chain Fv fragment and the light chain Fv fragment.
- the CH1 domain and CL domain of an antibody can be linked to each of the chain Fv fragment and the light chain Fv fragment.
- a peptide bond is attached to each C-terminus of the two light chain constant regions of the antibody structure or to each N-terminus of the light chain variable region. Via which each monovalent CD3 binding domain can be bound.
- bivalent scFv or sc via peptide bonds to each C terminus of two light chain constant regions or each N terminus of a light chain variable region. (Fv) 2 can be bound.
- each monovalent CD3 binding domain is attached to each N-terminus of two heavy chain variable regions of the antibody structure via a peptide bond.
- a bivalent scFv that is, sc (Fv) is connected to the N-terminus of one of the two heavy chain variable regions via a peptide bond.
- a bivalent scFv that is, only at the N-terminus of one heavy chain variable region of the two heavy chain variable regions of the antibody structure.
- Polypeptide aggregates to which sc (Fv) 2 binds are efficiently obtained.
- a monovalent scFv can be bound to the N-terminus of one heavy chain variable region of two heavy chain variable regions via a peptide bond.
- a monovalent scFv binds to the N-terminus of one of the heavy chain variable regions of the antibody structure.
- the polypeptide aggregate according to the present invention is efficiently obtained.
- each domain can be directly bonded by a peptide bond, and each domain can be bonded by a peptide bond via a peptide linker.
- the employed linker in addition to the linker exemplified in the above description, for example, a linker having a peptide tag such as His tag, HA tag, myc tag, and FLAG tag can be appropriately used.
- bonds together by a hydrogen bond, a disulfide bond, a covalent bond, an ionic interaction, or the combination of these bonds can also be utilized suitably.
- the affinity between CH1 and CL of an antibody is used, or the Fc region originating from the above-mentioned bispecific antibody is used for the association of hetero Fc regions.
- disulfide bonds formed between domains can also be suitably utilized.
- a structure that is a monovalent Fv and a monovalent Fab as an antigen-binding domain is also preferably used.
- VH and VL bound to the ends of heavy chain CH1 region and light chain CL region form an antibody binding domain by linking the other VL or VH fragment of monovalent Fv to the region via a peptide bond Structure to be used.
- an antibody binding domain and (3) sc (Fv) 2 forming a CD3 binding domain are linked via peptide bonds to the N-terminal of the other Fc region. obtain.
- a heavy chain CH1 region is present in one Fc region of the two Fc regions constituting the polypeptide aggregate, and the other.
- a polypeptide aggregate having a structure in which sc (Fv) 2 is linked to each Fc region via a peptide bond can be prepared.
- each domain can be bound by a peptide bond via a peptide linker in addition to being directly bound by a peptide bond.
- a linker having a peptide tag such as His tag, HA tag, myc tag, and FLAG tag can be appropriately used.
- a structure that is a bivalent scFv as an antigen-binding domain is also preferably used.
- one of the bivalent scFvs is (3) one of two Fc regions in which the binding activity to the Fc ⁇ receptor is reduced via VH constituting the CD3 binding domain.
- Two Fc regions that are linked to the Fc region by a peptide bond, and the other of the divalent scFvs is (3) decreased in binding activity to the Fc ⁇ receptor via VL constituting the CD3 binding domain.
- a polypeptide aggregate having a structure linked to the other Fc region of the above by a peptide bond can be produced.
- each domain can be bound by a peptide bond via a peptide linker in addition to being directly bound by a peptide bond.
- a linker having a peptide tag such as His tag, HA tag, myc tag, and FLAG tag can be appropriately used.
- a bivalent scFv is used as the antigen binding domain.
- one of the bivalent scFvs is (3) via an scFv constituting a CD3 binding domain;
- Two Fc regions with reduced binding activity are linked to one Fc region by a peptide bond, and the other of the bivalent scFvs is (2) two with reduced binding activity to Fc ⁇ receptors
- a polypeptide aggregate having a structure linked to the other Fc region of the Fc regions by a peptide bond can be produced.
- scFv constituting a CD3 binding domain is formed in one Fc region of two Fc regions constituting the polypeptide aggregate.
- a polypeptide assembly having a structure in which the scFv constituting the antigen binding domain is linked to the other Fc region via the peptide bond can be prepared.
- each domain can be bound by a peptide bond via a peptide linker in addition to being directly bound by a peptide bond.
- a linker to be employed a linker having a peptide tag such as a His tag, an HA tag, a myc tag, and a FLAG tag can be used as appropriate in addition to the linkers exemplified above.
- each of the antigen binding domain and the T cell receptor complex binding domain is a monovalent Fab is also preferably used.
- a heavy chain Fv fragment of a monovalent Fab constituting an antigen binding domain is linked to one polypeptide constituting an Fc region via a CH1 region, and the light chain Fv fragment of the Fab is a CL region.
- the heavy chain Fv fragment of the Fab constituting the T cell receptor binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region.
- Polypeptide aggregates having a structured structure can be produced.
- a heavy chain Fv fragment of a monovalent Fab constituting an antigen binding domain is linked to one polypeptide constituting an Fc region via a CH1 region, and the light chain Fv fragment of the Fab is The light chain Fv fragment of the Fab constituting the T cell receptor binding domain linked to the CL region is linked to the other polypeptide constituting the Fc region via the CH1 region, and the heavy chain Fv fragment of the Fab is the CL region.
- a polypeptide aggregate having a structure linked to can be produced.
- the heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is combined with the CL region.
- the light chain Fv fragment of the Fab constituting the antigen-binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the heavy chain Fv fragment of the Fab is linked to the CL region. Having polypeptide aggregates can also be made.
- a heavy chain Fv fragment of a monovalent Fab that constitutes an antigen-binding domain is linked to one polypeptide that constitutes an Fc region via a CH1 region, and the light chain Fv fragment of the Fab.
- the heavy chain Fv fragment of the Fab constituting the T cell receptor binding domain is linked to the other polypeptide constituting the Fc region via the CL region, and the light chain Fv fragment of the Fab is CH1.
- Polypeptide aggregates having structures linked to regions can be made.
- the heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is combined with the CL region.
- the antigen-binding domain and the T cell receptor complex-binding domain are each a monovalent Fab, which is another structure of the polypeptide aggregate according to the present invention
- a heavy chain Fv fragment having a monovalent Fab structure that binds to an antigen is linked to one polypeptide constituting the Fc region via a CH1 region, and a light chain Fv fragment having the Fab structure is linked to a CL region.
- An antigen-binding domain and (2) A heavy chain Fv fragment of monovalent Fab structure that binds to the T cell receptor complex is linked to the other polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab structure is A T cell receptor complex binding domain linked to a CL region, A heavy chain Fv fragment in the antigen binding domain and a light chain Fv fragment in the antigen binding domain or a heavy chain Fv fragment in the T cell receptor binding domain and a light chain Fv fragment in the T cell receptor binding domain Preferred examples thereof include polypeptides in which the charges of the CH1 region and CL region are controlled.
- the structure of the polypeptide aggregate is not limited to a specific structure.
- the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and the CL linked to the light chain Fv fragment in the antigen binding domain Polypeptide aggregates can be made in which the amino acid residues in the region have the same type of charge as each other.
- association control structure As another embodiment of the association control structure, the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the light chain Fv fragment in the T cell receptor complex binding domain Polypeptide aggregates in which the amino acid residues in the CL region have the same type of charge as each other can be produced.
- the amino acid residue of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and the light chain Fv fragment in the antigen binding domain have the same type of charge as each other, and the amino acid residues in the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the light chain Fv fragment in the T cell receptor complex binding domain Polypeptide aggregates in which the amino acid residues of the linked CL regions have the same type of charge as each other can be produced.
- association control structure it is linked to the CH1 region amino acid residue linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and to the light chain Fv fragment in the antigen binding domain.
- the amino acid residues in the CL region have the same type of charge as each other, and are linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and in the T cell receptor binding domain.
- a polypeptide aggregate in which the amino acid residues in the CL region linked to the light chain Fv fragment of the above have heterogeneous charges can be produced.
- the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and the light chain Fv fragment in the antigen binding domain have the same type of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the T cell receptor complex binding domain
- the amino acid residues of the CL region linked to the light chain Fv fragment have the same type of charge, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and the Polypeptide aggregates in which the amino acid residues in the CL region linked to the light chain Fv fragment in the T cell receptor binding domain have different charges from each other can be produced.
- the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the light chain Fv fragment in the T cell receptor complex binding domain are linked.
- the amino acid residues in the CL region have the same type of charge as each other, and are linked to the amino acid residues in the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and to the light chain Fv fragment in the antigen binding domain.
- polypeptide aggregates in which both amino acid residues in the CL region have different charges can be prepared.
- the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and the light chain Fv fragment in the antigen binding domain have the same type of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the T cell receptor complex binding domain
- the amino acid residues in the CL region linked to the light chain Fv fragment have the same type of charge, and the amino acid residues in the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the antigen binding domain
- Polypeptide aggregates in which both amino acid residues in the CL region linked to the light chain Fv fragment have different charges can be produced.
- the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and the light chain Fv fragment in the antigen binding domain have the same type of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the T cell receptor complex binding domain
- the amino acid residues of the CL region linked to the light chain Fv fragment have the same type of charge, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and the The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain, wherein the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor binding domain have different charges
- Polypeptide aggregates of amino acid residues of the CL regions linked in a chain Fv fragments have a heterogeneous charge each other
- control to inhibit association between the heavy chain of the T cell receptor binding domain and the light chain of the antigen binding domain and / or the heavy chain of the antigen binding domain and the light chain of the T cell receptor binding domain is possible to acquire a desired polypeptide aggregate molecule preferentially.
- the amino acid residue forming the interface between the heavy chain CH1 of the T cell receptor binding domain and the light chain CL of the antigen binding domain is changed to an amino acid residue having a positive charge
- the heavy chain CH1 of the antigen binding domain An example in which amino acid residues that form an interface between light chain CLs of the T cell receptor binding domain are changed to amino acid residues having a negative charge can be given.
- the association between the unintended T-cell receptor binding domain heavy chain CH1 and the antigen-binding domain light chain CL is inhibited because both amino acid residues forming the interface are positively charged and are not intended.
- the association between the heavy chain CH1 of the antigen binding domain and the light chain CL of the T cell receptor binding domain is inhibited because both amino acid residues forming the interface are negatively charged.
- the association between the heavy chain CH1 of the target T cell receptor binding domain and the light chain CL of the T cell receptor binding domain and the heavy chain CH1 of the target antigen binding domain and the light chain CL of the antigen binding domain can be efficiently obtained.
- the association of the heavy chain of the target T cell receptor binding domain and the light chain of the T cell receptor binding domain is promoted because the amino acid residues forming the interface have different charges from each other.
- the association of the heavy chain of the target antigen-binding domain and the light chain of the antigen-binding domain is also promoted because the amino acid residues forming the interface have different charges.
- the polypeptide aggregate of the present invention in which the target association has occurred can be efficiently obtained.
- CH1 T cell receptor binding domain heavy chain and antigen binding domain heavy chain
- CLs T cell receptor binding domain light chain and antigen
- -CH1 EU numbering at position 147 (eg, position 147 in the amino acid sequence of SEQ ID NO: 1) lysine (K) and relative (contacting) CL EU numbering at position 180 threonine (T) -CH1 EU numbering lysine at position 147 (K) and relative (contacting) CL EU numbering serine at position 131 (S) -CH1 EU numbering 147 position lysine (K) and relative (contacting) CL EU numbering position 164 position threonine (T) -CH1 EU numbering 147th lysine (K) and relative (contacting) CL EU numbering 138th asparagine (N) -CH1 EU numbering at position 147 lysine (K) and relative (contacting) CL EU numbering at position 123 glutamic acid (E) -CH1 EU numbering 175th glutamine (Q) and relative (contact) CL EU numbering 160th glutamine (Q) -CH1 EU numbering at position 213 lysine (K)
- EU numbering X position amino acid residue and “EU numbering X position amino acid” (X is an arbitrary number) are “amino number residue corresponding to EU numbering X position”, “EU numbering X position”. It can also be read as “amino acid corresponding to position”.
- a desired polypeptide aggregate can be obtained preferentially.
- amino acid residues are known to be highly conserved in humans and mice (J. Mol. Recognit. (2003) 16, 113-120).
- association of CH1 and CL the association of the constant region of the polypeptide aggregate of the present invention can be controlled by modifying the amino acid residue corresponding to the amino acid residue.
- the present invention relates to a polypeptide aggregate in which association between a heavy chain and a light chain is controlled, and one set selected from the group consisting of the following amino acid residue groups (a) to (f): Or a polypeptide aggregate in which two or more sets of amino acid residues have the same type of charge; (A) an amino acid residue contained in CH1 at the EU numbering position 147, and an amino acid residue contained in CL, the EU numbering position 180; (B) an amino acid residue contained in CH1, which is the amino acid residue at position 147 of EU numbering, and an amino acid residue contained in CL, which is an amino acid residue at position 131 in EU numbering; (C) an amino acid residue contained in CH1 at the EU numbering position 147, and an amino acid residue contained in CL, the EU numbering position 164; (D) an amino acid residue contained in CH1, which is the amino acid residue at position 147 of EU numbering, and an amino acid residue contained in CL, which is an amino acid residue at position 138 in EU numbering; (E)
- an antibody in which the amino acid residues in the set of amino acid residues shown in the following (g) are of the same kind; (G) An amino acid residue contained in CH1 and located at EU position 213, and an amino acid residue contained in CL and located at EU position 123.
- the “charged amino acid residue” is preferably selected from, for example, amino acid residues included in any of the following groups (X) or (Y); (X) glutamic acid (E), aspartic acid (D), (Y) Lysine (K), arginine (R), histidine (H).
- “having the same kind of charge” means, for example, that two or more amino acid residues are amino acid residues contained in any one group of (X) or (Y). It means having a group. “Having an opposite charge” means, for example, an amino acid residue in which at least one amino acid residue of two or more amino acid residues is included in any one group of the above (X) or (Y) Means that the remaining amino acid residues have amino acid residues contained in different groups.
- the present invention relates to a method for producing the above-mentioned polypeptide aggregate, and the present invention wherein the amino acid residues of the amino acid residue pairs shown in the above (a) to (g) are modified to be amino acid residues having the same kind of charge.
- This association control method is also a preferred embodiment of the present invention.
- amino acid residues to be used for “modification” are not limited to the amino acid residues in the constant region described above.
- a person skilled in the art can find amino acid residues that form an interface for polypeptide variants or heterologous multimers by homology modeling using commercially available software, etc. Amino acid residues can be subjected to modification.
- heavy chain variable region (VH) and light chain variable region (VL As an amino acid residue that contacts at the interface of), for example, glutamine (Q) at position 39 of the heavy chain variable region FR2 (for example, position 39 in the amino acid sequence described as SEQ ID NO: 6 in WO2006 / 106905), A glutamine (Q) at position 38 (for example, position 44 in the amino acid sequence described as SEQ ID NO: 8 in WO2006 / 106905) of the light chain variable region FR2 which is relative (contacted) may be mentioned.
- glutamine (Q) at position 39 of the heavy chain variable region FR2 for example, position 39 in the amino acid sequence described as SEQ ID NO: 6 in WO2006 / 106905
- a glutamine (Q) at position 38 for example, position 44 in the amino acid sequence described as SEQ ID NO: 8 in WO2006 / 106905
- leucine (L) at position 45 of the heavy chain variable region FR2 eg, position 45 in the amino acid sequence described as SEQ ID NO: 6 in WO2006 / 106905
- position 44 of the light chain variable region FR2 for example, A proline (P) at the 50th position in the amino acid sequence described in SEQ ID NO: 8 in WO2006 / 106905
- the numbering of these sites is based on Kabat et al. (Kabat EA et al. 1991.Sequence of Proteins of Immunological Interest. NIH).
- amino acid residues are known to be highly conserved in humans and mice (J. Mol. Recognit. (2003) 16, 113-120).
- the association of the variable region of the antibody can be controlled by modifying the amino acid residue corresponding to the amino acid residue.
- amino acid residues (1) and (2) or (3) and (4) have the same type of charge: Mention may be made of antibodies that are amino acid residues; (1) an amino acid residue contained in the heavy chain variable region and corresponding to position 39 of EU numbering; (2) an amino acid residue contained in the light chain variable region and corresponding to position 38 of EU numbering; (3) an amino acid residue contained in the heavy chain variable region and corresponding to the EU numbering position 45; (4) Amino acid residues contained in the light chain variable region and corresponding to EU numbering position 44.
- amino acid residues described in the above (1) and (2), (3) and (4) are close to each other when they are associated.
- a person skilled in the art can find a site corresponding to the amino acid residue described in the above (1) to (4) by homology modeling using a commercially available software for the desired heavy chain variable region or light chain variable region.
- the amino acid residue at the site can be subjected to modification.
- the “charged amino acid residue” is preferably selected from, for example, amino acid residues included in any of the following groups (X) or (Y); (X) glutamic acid (E), aspartic acid (D), (Y) Lysine (K), Arginine (R), Histidine (H).
- amino acid residues described in (1) to (4) above are generally (1) glutamine (Q), (2) Glutamine (Q), (3) Leucine (L), (4) Proline (P) It is. Therefore, in a preferred embodiment of the present invention, these amino acid residues are subjected to modification (for example, substitution with charged amino acids).
- the types of amino acid residues (1) to (4) are not necessarily limited to the above amino acid residues, and may be other amino acids corresponding to the amino acids.
- the amino acid corresponding to position 38 of EU numbering on the light chain variable region may be histidine (H), for example.
- H histidine
- the person skilled in the art refers to known literatures and the like (for example, J. Mol. Recognit. (2003) 16, 113-120) to determine the type of amino acid residue corresponding to the position for any position of the light chain.
- the amino acid residue can be appropriately modified (for example, substituted with a charged amino acid).
- the amino acid residue that can form a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) is, for example, opposite to leucine (L) at position 45 on the heavy chain variable region.
- a proline (P) at position 44 on the light chain variable region can be preferably exemplified.
- hydrophobic core refers to a part formed by aggregation of side chains of hydrophobic amino acids inside an associated polypeptide.
- Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine and the like.
- the formation of the hydrophobic core may involve amino acid residues other than hydrophobic amino acids (for example, tyrosine).
- This hydrophobic core serves as a driving force for advancing the association of water-soluble polypeptides together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside.
- hydrophobic amino acids of two different domains When hydrophobic amino acids of two different domains are present on the surface of the molecule and exposed to water molecules, entropy increases and free energy increases. Thus, the two domains associate with each other to reduce and stabilize the free energy, and the hydrophobic amino acids at the interface are buried inside the molecule, forming a hydrophobic core.
- hydrophobic amino acid that forms the hydrophobic core is changed to a charged polar amino acid, thereby inhibiting the formation of the hydrophobic core, resulting in inhibition of the polypeptide association. It is thought that.
- the “modification” of the present invention may facilitate the association of the first VH (VH1) and the first VL (VL1) and / or the second VH (VH2) and the second VL (VL2).
- the amino acid side chain present in the variable region of one H chain is replaced with a larger side chain (knob) and the amino acid side chain present in the opposite variable region of the other H chain is more
- the protrusions can be placed in the void to promote the association of VH1 and VL1, and / or VH2 and VL2, resulting in VH1 and VL2, and / or It is possible to further suppress the association between VH2 and VL1 polypeptides (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).
- each domain can be directly bonded by a peptide bond, and each domain can be bonded by a peptide bond via a peptide linker.
- a linker to be employed a linker having a peptide tag such as a His tag, an HA tag, a myc tag, and a FLAG tag can be used as appropriate in addition to the linkers exemplified above.
- bonds together by a hydrogen bond, a disulfide bond, a covalent bond, an ionic interaction, or the combination of these bonds can also be utilized suitably.
- the affinity between CH1 and CL of an antibody is used, or the Fc region originating from the above-mentioned bispecific antibody is used for the association of hetero Fc regions.
- disulfide bonds formed between domains can also be suitably utilized.
- polypeptide aggregate according to the present invention examples include the embodiments described in FIG. 17, FIG. 19, and FIG.
- polypeptide aggregate according to the present invention is produced by the same method as the method for producing the recombinant antibody.
- the present invention also relates to a polynucleotide encoding the polypeptide aggregate of the present invention.
- the polypeptide aggregate of the present invention can be incorporated into any expression vector.
- An appropriate host can be transformed with the expression vector to obtain a polypeptide aggregate-expressing cell.
- the polypeptide aggregate encoded by the polynucleotide can be obtained.
- the present invention relates to a vector comprising a polynucleotide encoding the polypeptide aggregate of the present invention, a cell carrying the vector, and a polypeptide comprising culturing the cell and recovering the polypeptide aggregate from the culture supernatant
- the present invention relates to a method for producing an aggregate. These can be obtained, for example, by the same technique as that for the recombinant antibody.
- the present invention provides a polymorph comprising (1) an antigen-binding domain, (2) a domain containing an Fc region with reduced Fc ⁇ receptor binding activity, and (3) a CD3-binding domain.
- a pharmaceutical composition containing a peptide aggregate as an active ingredient is provided.
- the present invention also relates to a therapeutic agent that induces cytotoxicity (cytotoxicity-inducing therapeutic agent), a cytostatic agent, and an anticancer agent that contains the aggregate as an active ingredient.
- the pharmaceutical composition of the present invention can also be used as a cancer therapeutic agent or cancer preventive agent.
- the cytotoxicity-inducing therapeutic agent, cytostatic agent and anticancer agent of the present invention are preferably administered to a subject suffering from cancer or a subject who is likely to relapse.
- an antigen binding domain (2) a domain containing an Fc region with reduced Fc ⁇ receptor binding activity, and (3) a CD3 binding domain
- a cytotoxic injury-inducing therapeutic agent, a cytostatic agent and an anticancer agent which comprise a polypeptide aggregate containing the above as an active ingredient, a method for preventing or treating cancer comprising the step of administering the polypeptide aggregate to a subject, or It can also be expressed as the use of the polypeptide aggregate in the production of a cytotoxicity-inducing therapeutic agent, a cytostatic agent and an anticancer agent.
- a polypeptide aggregate containing “(1) an antigen-binding domain, (2) a domain containing an Fc region with reduced Fc ⁇ receptor binding activity, and (3) a CD3 binding domain as an active ingredient” “Contains” means that the polypeptide aggregate is included as a main active ingredient, and does not limit the content of the polypeptide aggregate.
- a plurality of types of polypeptide aggregates can be blended with the pharmaceutical composition or the cytotoxicity-inducing therapeutic agent, the cell growth inhibitor and the anticancer agent according to the present invention, if necessary.
- a cocktail of a plurality of polypeptide aggregates of the present invention that bind to the same antigen there is a possibility that the cytotoxic action against cells expressing the antigen can be enhanced.
- the therapeutic effect can be improved by formulating the polypeptide assembly of the present invention containing an antigen-binding domain that binds to another cancer antigen. Enhanced.
- the polypeptide aggregate of the present invention is encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin and poly [methylmethacrylic acid]), and colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, may be a nano-particles, and nano-capsules) ( "Remington's Pharmaceutical Science 16 th edition", Oslo Ed. (1980) see the like).
- microcapsules such as hydroxymethylcellulose, gelatin and poly [methylmethacrylic acid]
- colloid drug delivery systems liposomes, albumin microspheres, microemulsions, may be a nano-particles, and nano-capsules
- a method of making a drug a sustained-release drug is also known, and this method can be applied to the polypeptide aggregate of the present invention (J. Biomed. Mater. Res. (1981) 15, 267-277, Chemtech (1982) 12, 98-105, US Pat. No. 3,773,719, European
- the pharmaceutical composition of the present invention, or the cell growth inhibitor and the anticancer agent can be administered to a patient by either oral or parenteral administration. Preferably, it is parenteral administration. Specific examples of such administration methods include injection administration, nasal administration, transpulmonary administration, and transdermal administration. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection.
- the pharmaceutical composition of the present invention, or the cytotoxicity-inducing therapeutic agent, cytostatic agent and anticancer agent can be administered systemically or locally by injection administration.
- the administration method can be appropriately selected depending on the age and symptoms of the patient. As the dose, for example, the dose can be selected in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, for example, the dose can be selected in the range of 0.001 mg / body to 100,000 mg / body per patient. However, the pharmaceutical composition of the present invention is not limited to these doses.
- the pharmaceutical composition of the present invention can be formulated in accordance with a conventional method (for example, Remington's Pharmaceutical, Science, Latest Edition, Mark Publishing, Company, Easton, USA) together with pharmaceutically acceptable carriers and additives. It may be.
- a conventional method for example, Remington's Pharmaceutical, Science, Latest Edition, Mark Publishing, Company, Easton, USA
- pharmaceutically acceptable carriers and additives may be.
- it is not limited to these, and other commonly used carriers can be used as appropriate.
- silicic acid lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride
- the carrier include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts.
- the present invention provides a method for causing injury to cells expressing an cancer antigen by suppressing the expression of the cancer antigen by contacting the cell expressing the cancer antigen with the polypeptide aggregate of the present invention that binds to the cancer antigen.
- the monoclonal antibody that binds to the cancer antigen is as described above as the polypeptide aggregate of the present invention that binds to the cancer antigen contained in the cytotoxicity-inducing therapeutic agent, cytostatic agent and anticancer agent of the present invention.
- the cell to which the polypeptide aggregate of the present invention that binds to the cancer antigen binds is not particularly limited as long as the cell expresses the cancer antigen.
- preferable cancer antigen-expressing cells in the present invention include ovarian cancer, prostate cancer, breast cancer, uterine cancer, liver cancer, lung cancer, pancreatic cancer, stomach cancer, bladder cancer, and colon cancer cells.
- the cancer antigen is GPC3, it is not limited as long as it is a cancer cell expressing GPC3.
- suitable cancer cells include hepatocellular carcinoma, lung cancer, ovarian cancer and the like.
- “contact” is performed, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to a culture solution of cancer antigen-expressing cells cultured in a test tube.
- a shape of the added polypeptide aggregate a shape such as a solid obtained by solution or lyophilization can be appropriately used.
- an aqueous solution it may be an aqueous solution containing only the polypeptide aggregate of the present invention purely.
- the surfactant, excipient, coloring agent, flavoring agent, preservative described above may also be a solution containing a stabilizer, buffer, suspending agent, tonicity agent, binder, disintegrant, lubricant, fluidity promoter, flavoring agent, and the like.
- concentration to be added is not particularly limited, but the final concentration in the culture medium is preferably in the range of 1 pg / ml to 1 / g / ml, more preferably 1 ng / ml to 1 mg / ml, and still more preferably 1 ⁇ g / ml to 1 mg / ml can be suitably used.
- “contact” may be administered to a non-human animal transplanted with cancer antigen-expressing cells in the body or an animal having cancer cells that endogenously express the cancer antigen in another embodiment.
- the administration method can be carried out by either oral or parenteral administration. Particularly preferred is an administration method by parenteral administration, and specific examples of the administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection.
- the pharmaceutical composition of the present invention, or the cytotoxicity-inducing therapeutic agent, cell growth inhibitor and anticancer agent can be administered systemically or locally by injection administration.
- the administration method can be appropriately selected depending on the age and symptoms of the test animal.
- it When it is administered as an aqueous solution, it may be an aqueous solution containing purely the polypeptide aggregate of the present invention.
- the surfactant, excipient, colorant, flavoring agent, storage described above It may be a solution containing a lubricant, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrant, a lubricant, a fluidity promoter, a corrigent and the like.
- the dose for example, the dose can be selected in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient.
- the polypeptide aggregate dose of the present invention is not limited to these doses.
- the following method is preferably used as a method for evaluating or measuring cytotoxicity caused by cells expressing an antigen to which an antigen-binding domain constituting the polypeptide aggregate binds by contacting the polypeptide aggregate of the present invention. Is done.
- methods for evaluating or measuring the cytotoxic activity in vitro include methods for measuring cytotoxic T cell activity. Whether or not the polypeptide aggregate of the present invention has T-cell cytotoxic activity can be measured by a known method (for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993)).
- a polypeptide aggregate that binds to an antigen that binds to an antigen whose antigen-binding domain is different from that of the present invention and that is not expressed by the cells used in the test is used.
- the activity can be determined by showing a stronger cytotoxic activity of the polypeptide aggregate of the present invention than the polypeptide aggregate used as a control.
- cells expressing an antigen to which the antigen-binding domain constituting the polypeptide aggregate of the present invention binds are intradermally or subcutaneously in a non-human test animal. After transplantation, the test polypeptide aggregate is administered intravenously or intraperitoneally every day or every several days from that day or the next day. By measuring the tumor size over time, the difference in change in the tumor size can be defined as cytotoxic activity.
- a control polypeptide aggregate was administered, and the tumor size in the group administered with the polypeptide aggregate of the present invention was more significant than the tumor size in the control polypeptide aggregate administration group. Can be determined to have cytotoxic activity.
- an isotope-labeled thymidine cell is applied as a method for evaluating or measuring the inhibitory effect on the proliferation of a cell expressing an antigen to which the antigen-binding domain constituting the polypeptide aggregate binds by contacting the polypeptide aggregate of the present invention.
- Uptake measurement and MTT method are preferably used.
- the same method as the method for evaluating or measuring the cytotoxic activity in vivo as described above can be suitably used as a method for evaluating or measuring the cell growth inhibitory activity in vivo.
- the present invention also provides a kit for use in the method of the present invention, comprising the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention.
- the kit can be packaged with pharmaceutically acceptable carriers, media, instructions describing the method of use, and the like.
- the present invention also relates to the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention for use in the method of the present invention.
- ERY2 a molecule in which an Fc region (silent Fc) with reduced Fc ⁇ receptor binding activity was linked to BiTE via a polypeptide linker, was prepared, and this activity was compared with BiTE.
- BiTE against GPC3 is linked by linking the scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon with a short peptide linker.
- GPC3 BiTE was produced (FIG. 17A).
- ERY2 GPC3 ERY2
- FIG. 17C ERY2 for GPC3, to which silent type Fc was linked
- the IgG type anti-GPC3 antibody was prepared as an antibody having a reduced fucose content in the sugar chain part, which is known to further enhance ADCC activity, that is, a low fucose type antibody.
- oligonucleotides have a base sequence that encodes a partial sequence of the H chain variable region (M12HVH) and L chain variable region (M12 VL) of the anti-CD3 antibody (M12), and its terminal sequence has a complementary sequence.
- Oligonucleotides were made. These series of oligonucleotides are linked via their complementary sequence by polymerase reaction, and designed to synthesize polynucleotides corresponding to the H chain variable region (M12 VH) and L chain variable region (M12 VL). It was done. After the oligonucleotides were mixed, these oligonucleotides were linked by a PCR method, and two cDNAs encoding the amino acid sequences of each variable region were obtained.
- a primer consisting of a sequence in which M12 ⁇ VL and M12 VH repeat Gly, Gly, Gly, Gly, and Ser (SEQ ID NO: 7) three times by PCR using primers with appropriate sequences and these cDNAs as templates.
- anti-GPC3 scFv and M12 scFv were converted to Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Gly, Amino acids linked by a linker consisting of a Ser sequence (SEQ ID NO: 7) and having a His tag (8 Hiss) added to the C-terminus thereof (except for 19 amino-terminal amino acids described in SEQ ID NO: 33) A cDNA fragment encoding the sequence was generated.
- a cDNA fragment was prepared in which a nucleotide sequence encoding a kozac sequence and a secretory signal sequence was added, and a NotI cleavage site was added on the 3 ′ side thereof.
- GPC3 BiTE SEQ ID NO: 33, the amino terminal 19 amino acids as the signal sequence is not included in the mature sequence
- the vector was introduced into the CHO cell line DG44 by electroporation.
- Drug-resistant cell lines were isolated by culturing the transfected cells in the presence of 1 mg / mL Geneticine after limiting dilution.
- a cell line expressing GPC3 BiTE was selected by performing Western blot analysis on the culture supernatant of the obtained cell line using an antibody against the His tag.
- the culture supernatant obtained by culturing a large amount of the cell line was added to an SP Sepharose FF column (GE Healthcare). After washing the column, the fraction containing GPC3 BiTE was eluted with a NaCl concentration gradient. Further, the fraction was added to a HisTrap HP column (GE Healthcare). After washing the column, a fraction containing GPC3 BiTE was eluted with a concentration gradient of imidazole. After the fraction was concentrated with an ultrafiltration membrane, the concentrated solution was added to a Superdex® 200 column (GE® Healthcare). By collecting only the monomeric GPC3 BiTE fraction, purified GPC3 BiTE was obtained.
- GPC3 ERY2 Methods known to those skilled in the art, such as PCR using primers to which appropriate sequences similar to those described above were added, and methods using QuikChange Site-Directed Mutagenesis Kit (Stratagene) GPC3 ERY2_Hk (SEQ ID NO: 34, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequence), and GPC3 ERY2_Hh (SEQ ID NO: 35, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequences) Expression vectors into which the polynucleotides encoding each (not included) were inserted were generated.
- GPC3 ERY2_Hk SEQ ID NO: 34, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequence
- GPC3 ERY2_Hh SEQ ID NO: 35, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequences
- GPC3 ERY2 was transiently expressed.
- the obtained culture supernatant was added to an Anti-FLAG-M2 column (Sigma). After washing the column, elution with 0.1 mg / mL-FLAG peptide (Sigma) was performed.
- the fraction containing GPC3 ERY2 was added to a HisTrap HP column (GE Healthcare), and the column was washed and then eluted with a concentration gradient of imidazole.
- the mononuclear cell fraction layer was collected. After the cells of the mononuclear cell fraction were washed once with Dulbecco's Modified Eagle's Medium (SIGMA, 10% FBS / D-MEM) containing 10% FBS, the cells were treated with 10% FBS / D-MEM. The cell density was adjusted to 4 ⁇ 10 6 / mL. The cell solution thus prepared was used as a human PBMC solution in the subsequent tests.
- SIGMA Dulbecco's Modified Eagle's Medium
- cytotoxic activity was evaluated based on the cell growth inhibition rate using a xCELLigence real-time cell analyzer (Roche Diagnostics).
- the SK-pca13a cell line established by forcibly expressing human GPC3 in the SK-HEP-1 cell line was used as the target cell.
- SK-pca13a is detached from the dish, seeded at 100 ⁇ L / well on an E-Plate 96 (Roche Diagnostics) plate at 1 ⁇ 10 4 cells / well, and live cells using xCELLigence real-time cell analyzer Measurement was started.
- the plate was taken out from the xCELLigence real-time cell analyzer, and 50 ⁇ L of each antibody prepared at each concentration (0.004, 0.04, 0.4, 4 nM) was added to the plate. After reacting at room temperature for 15 minutes, 50 ⁇ L (2 ⁇ 10 5 cells / well) of the human PBMC solution prepared in (5-1) was added, and the plate was re-set in the xCELLigence real-time cell analyzer. Cell measurement started. The reaction was carried out under the conditions of 5% carbon dioxide gas and 37 ° C., and the cell growth inhibition rate (%) was determined from the Cell Index value 72 hours after addition of human PBMC by the following formula. As the Cell Index value used for the calculation, a numerical value after normalization was used so that the Cell Index value immediately before the addition of the antibody was 1.
- A indicates the average Cell Index value in the wells to which no antibody was added (target cells and human PBMC only), and B indicates the average Cell Index value in each well.
- the test was conducted in triplicate.
- GPC3 BiTE When the cytotoxic activity of GPC3 ⁇ BiTE, GPC3 ERY2, and IgG-type GPC3 antibody was measured using PBMC (Peripheral Blood Mononuclear Cell) prepared from human blood as effector cells, GPC3 BiTE showed extremely strong activity (Fig. 1). ). This activity was much stronger than that of the low fucose type anti-GPC3 antibody, and it was considered that GPC3 ⁇ ⁇ BiTE could be an excellent cancer therapeutic agent that surpasses IgG type antibody. On the other hand, GPC3 ERY2 showed activity higher than that of IgG anti-GPC3 antibody, but did not reach the activity of GPC3 BiTE. This suggests that the target molecule cannot be created simply by adding Fc to BiTE.
- PBMC Peripheral Blood Mononuclear Cell
- Target molecule GPC3 ERY5 Polypeptide encoded by the polynucleotide inserted in the expression vector: GPC3 ERY5_Hh (SEQ ID NO: 36, the amino terminal 19 amino acids as the signal sequence is not included in the mature sequence), GPC3 ERY2_Hk
- GPC3 ERY6 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 ERY6_Hk (SEQ ID NO: 37, the amino terminal 19 amino acids as the signal sequence is not included in the mature sequence), GPC3 ERY6_Hh (SEQ ID NO: 38, signal The amino terminal 19 amino acids that are sequences are not included in the mature sequence)
- Target molecule GPC3 ERY7 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 ERY7_Hh (SEQ ID NO: 39, the amino terminal 19 amino acids as the signal sequence is not included in the mature sequence), GPC3 ERY7_L (SEQ ID NO: 40, signal The amino terminal 19 amino acid sequence is not included in the mature sequence), GPC3 ERY2_Hk
- the obtained culture supernatant was added to an Anti-FLAG-M2 column (Sigma). After washing the column, elution with 0.1 mg / mL-FLAG peptide (Sigma) was performed.
- the fraction containing the target molecule was added to a HisTrap HP column (GE Healthcare), and the column was washed and then eluted with an imidazole concentration gradient. After the fraction containing the target molecule was concentrated with an ultrafiltration membrane, the fraction was added to a Superdex 200 column (GE Healthcare) and purified by collecting only the monomer fraction of the eluate. Each target molecule was obtained.
- GPC3 ERY8-2_Hk (SEQ ID NO: 41, the amino terminal 19 amino acid as the signal sequence is matured by a method known to those skilled in the art such as PCR using a primer added with an appropriate sequence similar to the method described above.
- GPC3 ERY8-2_Hh (SEQ ID NO: 42, the amino terminal 19 amino acids as the signal sequence are not included in the mature sequence)
- GPC3 ERY9-1_H (SEQ ID NO: 43, the signal sequence) 19 amino acids at the amino terminal are not included in the mature sequence
- GPC3 ERY9-1_ L-His (SEQ ID NO: 44, the amino acid at the amino terminal 19 which is the signal sequence is not included in the mature sequence
- GPC3 ERY9-1_ L -FLAG (SEQ ID NO: 45, amino terminal 19 amino acids as signal sequence is not included in mature sequence)
- GPC3 ERY10-1_Hh (SEQ ID NO: 46, amino terminal 19 amino acids as signal sequence)
- a series of expression vectors which polynucleotide has been inserted for each encoding non) included in the mature sequence was produced.
- GPC3 ERY8-2 Polypeptide encoded by polynucleotide inserted into expression vector: GPC3 ERY8-2_Hk (SEQ ID NO: 41, amino terminal 19 amino acids as signal sequence is not included in mature sequence), GPC3 ERY8-2_Hh (SEQ ID NO: : 42, the amino terminal 19 amino acids which are signal sequences are not included in the mature sequence), GPC3 ERY7_L
- GPC3 ERY9-1 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 ERY9-1_H (SEQ ID NO: 43, the amino terminal 19 amino acids as the signal sequence is not included in the mature sequence), GPC3 ERY9-1_L-His ( SEQ ID NO: 44, amino terminal 19 amino acids that are signal sequences are not included in the mature sequence), GPC3 ERY9-1_L-FLAG (SEQ ID NO: 45, amino terminal 19 amino acids that are signal sequences are included in the mature sequences) Absent)
- Target molecule GPC3 ERY10-1 Polypeptide encoded by polynucleotide inserted in expression vector: GPC3 ERY10-1_Hh (SEQ ID NO: 46, amino terminal 19 amino acids as signal sequence is not included in mature sequence), GPC3 ERY8-2_Hk, GPC3 ERY7_L
- the obtained culture supernatant was added to an Anti-FLAG-M2 column (Sigma), and the column was washed and then eluted with 0.1 mg / mL-FLAG peptide (Sigma).
- the fraction containing the target molecule was added to a HisTrap HP column (GE Healthcare), and the column was washed and then eluted with an imidazole concentration gradient. After the fraction containing the target molecule was concentrated by ultrafiltration, the fraction was added to a Superdex 200 column (GE Healthcare) and purified by collecting only the monomer fraction of the eluate. The target molecule was obtained.
- GPC3 ERY9-1 and GPC3 ERY10-1 showed significantly stronger cytotoxic activity than BiTE in molecules with a large distance between the binding domain for cancer antigen and the binding domain for CD3 epsilon. It was a result.
- NK cells were removed from PBMCs isolated from blood collected from healthy volunteers using CD56 MicroBeads, human (MCAS Miltenyi biotec).
- Human lung squamous cell carcinoma cell line PC-10 (Immunobiology Laboratories) 5 ⁇ 10 6 cells, human PBMC 4.5 ⁇ 10 6 cells from which NK cells were removed, and Matrigel basement membrane matrix (BD) were mixed.
- NOD scid mice (CLEA Japan, female, 7W) were transplanted subcutaneously at the groin. The day of transplantation was defined as day 0.
- anti-asialo GM1 antibody (Wako Pure Chemical Industries) was intraperitoneally administered to mice at 0.2 mg / mouse.
- GPC ERY8-2 was intraperitoneally administered at 30 ⁇ g / mouse.
- GPC ERY8-2 was administered 5 times from day 0 to 4.
- NK cells were removed from PBMCs isolated from blood collected from healthy volunteers using CD56 MicroBeads, human (MACS Miltenyi biotec).
- Human lung squamous cell carcinoma cell line PC-10 (Immuno-Biological Laboratories) 5 ⁇ 10 6 cells, human PBMC 4.5 ⁇ 10 6 cells from which NK cells have been removed, and Matrigel basement membrane matrix (BD) are mixed NOD scid mice (CLEA Japan, female, 7W) were transplanted subcutaneously at the groin. The day of transplantation was defined as day 0.
- anti-asialo GM1 antibody (Wako Pure Chemical Industries) was intraperitoneally administered to mice at 0.2 mg / mouse.
- GPC ERY10-1 was intraperitoneally administered at 30 ⁇ g / mouse.
- GPC ERY10-1 was administered 13 times in total from day 0 to 4, day 7 to 11, and day 14 to 16.
- in-vivo drug efficacy was evaluated by GPC3 ERY10-1. That is, T cells proliferated by culturing human PBMC in vitro were transferred to NOD scid mice in which tumor formation by the transplanted PC-10 was confirmed. The mice were treated with GPC3 ERY10-1 (referred to as a T cell transfer model).
- mice On the day before transplantation and on days 6, 8, 12, 16, and 20, the mice were intraperitoneally administered with 0.2 mg / animal of anti-asialo GM1 antibody (Wako Pure Chemical Industries). On the 6th day after transplantation, grouping was performed according to tumor size and body weight, and then T cells obtained by the expansion culture were transplanted intraperitoneally at 1 ⁇ 10 7 cells / mouse. Two hours later, GPC ERY10-1 was intraperitoneally administered at 30 ⁇ g / animal. GPC ERY10-1 was administered five times on days 7, 8, 12, 16, and 17.
- GPC3 ERY9-1 and GPC3 ERY10-1 were administered at 30 ⁇ g / mouse into the abdominal cavity of NOD scid mice (Claire Japan, female, 8W). After administration, blood was collected from the buccal vein of mice at 15 min, 2 hours, 1 day, 2 days, and 7 days, using hematocrit capillaries (Terumo), and the plasma was prepared.
- GPC3 ERY9-1 and GPC3 ERY10-1 were added to GPC3 ERY9 and GPC3 ERY9-1 as appropriate to Ba / F3 cells expressing GPC3 (GPC3 / BaF) and Ba / F3 cells expressing human CD3 epsilon (CD3 / BaF).
- -1 or GPC3 ERY10-1 was reacted with GPC3 / BaF or CD3 / BaF. After washing these cells, a FITC-labeled secondary antibody was added, and the secondary antibody was further reacted. After washing the cells, the fluorescence intensity labeled on the cells was measured with an Epis® XL flow cytometer (Beckman® coulter), and a standard curve was prepared for each antibody.
- Plasma prepared from blood collected over time from mice administered with GPC3-ERY9-1 and GPC3-ERY10-1 was diluted as appropriate.
- the plasma is reacted with GPC3 / BaF and CD3 / BaF in the same manner as the standard curve above, and the amount of GPC3 ⁇ ERY9-1 and GPC3 ERY10-1 present in the plasma is measured. It was. Using the measured value and the standard curve, the concentration of each antibody in plasma was calculated.
- GPC315ERY15- by a method known to those skilled in the art such as a PCR method using a primer to which an appropriate sequence is added and a method using QuikChange Site-Directed Mutagenesis Kit (Stratagene).
- 1_Hh (SEQ ID NO: 47, amino sequence 19 amino acids which are signal sequences are not included in the mature sequence)
- GPC3 ERY15-1_Hk (SEQ ID NO: 48, amino sequence 19 amino acids which are the signal sequences are included in the mature sequences)
- Expression vectors into which the respective polynucleotides encoding each were inserted were prepared.
- GPC3 ERY15-1_Hh (SEQ ID NO: 47, amino acid terminal 19 amino acid as signal sequence is not included in mature sequence)
- GPC3 ERY15-1_Hk (SEQ ID NO: 48, amino acid terminal 19 amino acid as signal sequence is included in mature sequence)
- GPC3 ERY7_L expression vectors were introduced into FreeStyle293-F cells to express GPC3 ERY15-1 transiently.
- the obtained culture supernatant was added to an Anti-FLAG-M2 column (Sigma). After washing the column, elution with 0.1 mg / mL-FLAG peptide (Sigma) was performed.
- the fraction containing GPC3 ERY15-1 was added to a HisTrap HP column (GE Healthcare), and the column was washed and then eluted with a concentration gradient of imidazole. After the fraction containing GPC3 ERY15-1 was concentrated by ultrafiltration, the fraction was added to a Superdex 200 column (GE Healthcare), collecting only the GPC3 ERY15-1 fraction of the eluent monomer As a result, purified GPC3 ERY15-1 was obtained.
- GPC3 ERY15-1 has a cancer antigen-independent cytokine-inducing ability compared with that of GPC3 BiTE, GPC3 ERY9-1, GPC3 ERY10-1, and catumaxomab It was.
- PBMCs were prepared from blood collected from healthy volunteers by the method described above. 50 ⁇ L of each antibody prepared to 40 nM was added to 50 ⁇ L of human PBMC solution (2 ⁇ 10 5 cells / well), and 100 ⁇ L of 10% FBS / D-MEM was further added. The reaction solution was cultured under conditions of 5% carbon dioxide and 37 ° C.
- the culture supernatant was collected, and the cytokine secreted into the culture supernatant was quantified by the Cytomometric Beads Array (CBA) method using Human Th1 / Th2 / Th17 Kit (BD). .
- CBA Cytomometric Beads Array
- BD Human Th1 / Th2 / Th17 Kit
- GPC3 ERY15-1 with FcgR-binding Fc and catumaxomab showed clear cytokine induction
- GPC3 BiTE without Fc and GPC3 ERY9-1 showed clear cytokine induction
- GPC3 ERY10 with silent Fc No cytokine induction was observed with -1 (FIG. 11). Therefore, a series of molecules such as GPC3GERY8-2, GPC3 ERY9-1, GPC3 ERY10-1 etc. with silent Fc do not cause cancer antigen-independent cytokine induction and are very safe molecules It was considered.
- GPC3 ERY18 L1, L2, L3, L4, S1 A molecule having a CD3 binding domain different from the scFv structure was examined.
- GPC3 ERY18 (FIG. 17K), which is a molecular type in which the VH region and VL region of the CD3 antibody were bound to the C terminus of the two H chains of IgG against the cancer antigen (GPC3), was prepared.
- GPC3 ERY18 L1 to L4 were prepared in which the number of linkers (Gly, Gly, Gly, Gly, and Ser) was changed from 1 to 4.
- a molecule (GPC3 ERY18 S1) that allows introduction of a disulfide bond by substituting an amino acid at an appropriate site with Cys was also produced.
- GPC3 ERY18 L1_Hh (SEQ ID NO: 49, the amino terminal 19 amino acid as a signal sequence is a mature sequence) by a method known to those skilled in the art such as PCR using a primer added with an appropriate sequence in the same manner as described above.
- GPC3 ERY18 L1_Hk (SEQ ID NO: 50, signal sequence amino terminal 19 amino acids are not included in the mature sequence)
- GPC3 ERY18 L2_Hh (SEQ ID NO: 51, signal sequence amino terminal 19) Amino acids are not included in the mature sequence)
- GPC3 ERY18 L2_Hk (SEQ ID NO: 52, the amino terminal 19 amino acids of the signal sequence are not included in the mature sequence)
- GPC3 ERY18 L3_Hh (SEQ ID NO: 53, in the signal sequence)
- Some amino terminal 19 amino acids are not included in the mature sequence
- GPC3 ERY18 L3_Hk (SEQ ID NO: 54, amino terminal 19 amino which is the signal sequence) Is not included in the mature sequence)
- GPC3 ERY18 L4_Hh (SEQ ID NO: 55, signal sequence amino terminal 19 amino acids are not included in the mature sequence)
- GPC3 ERY18 L4_Hk (SEQ ID NO: 56, signal sequence)
- G. Target molecule GPC3 ERY18 L1 Expression vector: GPC3 ERY18 L1_Hh (SEQ ID NO: 49, amino terminal 19 amino acid as signal sequence is not included in mature sequence), GPC3 ERY18 L1_Hk (SEQ ID NO: 50, amino terminal 19 amino acid as signal sequence is mature sequence) Not included) and GPC3 ERY7 L
- GPC3 ERY18 L2 Expression vector: GPC3 ERY18 L2_Hh (SEQ ID NO: 51, amino terminal 19 amino acid as signal sequence is not included in mature sequence), GPC3 ERY18 L2_Hk (SEQ ID NO: 52, amino terminal 19 amino acid as signal sequence is mature sequence) Not included) and GPC3 ERY7 L
- Target molecule GPC3 ERY18 L3
- GPC3 ERY18 L3_Hh SEQ ID NO: 53, amino acid terminal 19 amino acid as signal sequence is not included in mature sequence
- GPC3 ERY18 L3_Hk SEQ ID NO: 54, amino acid terminal 19 amino acid as signal sequence is mature sequence
- GPC3 ERY18 L4 Expression vector: GPC3 ERY18 L4_Hh (SEQ ID NO: 55, amino terminal 19 amino acid as signal sequence is not included in mature sequence), GPC3 ERY18 L4_Hk (SEQ ID NO: 56, amino terminal 19 amino acid as signal sequence is mature sequence) Not included) and GPC3 ERY7 L
- Target molecule GPC3 ERY18 S1
- Expression vector GPC3 ERY18 S1_Hh (SEQ ID NO: 57, amino terminal 19 amino acid as signal sequence is not included in mature sequence)
- GPC3 ERY18 S1_Hk SEQ ID NO: 58, amino terminal 19 amino acid as signal sequence is mature sequence Not included
- the obtained culture supernatant was added to an Anti-FLAG-M2 column (Sigma), and the column was washed and then eluted with 0.1 mg / mL-FLAG peptide (Sigma).
- the fraction containing the target molecule was added to a HisTrap HP column (GE Healthcare), and the column was washed and then eluted with an imidazole concentration gradient. After the fraction containing the target molecule was concentrated by ultrafiltration, the fraction was added to a Superdex 200 column (GE Healthcare) and purified by collecting only the monomer fraction of the eluate. The target molecule was obtained.
- GPC3 ERY19-3 a molecular type in which the VH and CH1 regions of the CD3 antibody, and the VL and CL regions were bound to the C-terminals of the two H chains of the IgG antibody against the cancer antigen (GPC3), respectively, was prepared ( FIG. 17L). That is, GPC3 ERY19-3_Hh (SEQ ID NO: 59, the amino terminal 19 amino acid as the signal sequence is matured by a method known to those skilled in the art, such as PCR using a primer added with an appropriate sequence in the same manner as described above. Expression vector in which polynucleotides encoding GPC3 ERY19-3_Hk (SEQ ID NO: 60, signal sequence amino-terminal 19 amino acids are not included in the mature sequence) are inserted. It was.
- GPC3 ERY19-3_Hh (SEQ ID NO: 59, amino acid terminal 19 amino acid as signal sequence is not included in mature sequence)
- GPC3 ERY19-3_Hk (SEQ ID NO: 60, amino acid terminal 19 amino acid as signal sequence is included in mature sequence)
- GPC3 ERY7_L expression vectors were introduced into FreeStyle293-F cells to transiently express GPC3 ERY19-3.
- the obtained culture supernatant was added to a HiTrap rProtein A FF column (GE Healthcare), and the column was washed and then eluted with an acid.
- Example 6 Preparation of polypeptide aggregates using only the protein A purification step by introducing mutations into the CH3 domain of GPC3 ERY 10-1
- GPC3 ERY10-1 prepared in Example 3 CH3 Knobs-into-hole is used as the domain structure. A His tag and a FLAG tag are added to the C-terminus of each H chain. By performing two types of affinity purification using these tags, GPC3 in which the two types of target H chains are hetero-associated The ERY10-1 molecule was purified.
- a polypeptide aggregate having an Fc domain is first purified from culture supernatant of cells expressing GPC3 ERY10-1 using protein A chromatography.
- GC33 (2) H anti-human Glypican-3 antibody heavy chain variable region, SEQ ID NO: 61, amino terminal 19 which is a signal sequence
- a gene encoding (amino acids are not included in the mature sequence) was generated by methods known to those skilled in the art.
- a gene encoding GC33-k0 anti-human Glypican-3 antibody L chain, SEQ ID NO: 62, the amino terminal 19 amino acids as the signal sequence is not included in the mature sequence
- an antibody H chain constant region the following genes were prepared by methods known to those skilled in the art.
- L. Target molecule LALA-G1d LALA-G1d (sequence where EU numbering 234th and 235th Leu in IgG1 sequence was replaced with Ala, 297th Asn was replaced with Ala, and Cly terminal Gly and Lys were removed. (Number: 63, the amino terminal 19 amino acids as the signal sequence are not included in the mature sequence)
- LALA-G1d-CD3 CD3 scFv anti-human CD3 antibody H chain variable region and anti-human CD3 antibody L chain variable region is polypeptide
- LALA-G1d SEQ ID NO: 63, amino acid terminal 19 amino acid which is a signal sequence is not included in the mature sequence
- LALA-G1d-CD3 SEQ ID NO: 64, signal sequence amino acid 19 amino acids are not included in the mature sequence
- Target molecule LALA-G3S3E-G1d Mutation that replaces EU numbering 435th His with Arg in the sequence of LALA-G1d (SEQ ID NO: 63, amino acid terminal 19 amino acid which is a signal sequence is not included in the mature sequence) and EU numbering 439th Lys LALA-G3S3E-G1d introduced with a mutation to substitute for Glu (SEQ ID NO: 65, the amino terminal 19 amino acids which are signal sequences are not included in the mature sequence)
- Target molecule LALA-S3K-G1d-CD3 LALA-G1d-CD3 (SEQ ID NO: 64, 19 amino acids at the amino terminal of the signal sequence is not included in the mature sequence) was introduced with a mutation that replaces the 356th Asp of EU numbering with Lys. S3K-G1d-CD3 (SEQ ID NO: 66, the amino terminal 19 amino acids which are signal sequences are not included in the mature sequence).
- Anti-human GPC3 antibody H chain gene NTA1L or NTA1R was prepared by linking LALA-G1d-CD3 or LALA-G1d downstream of GC33 (2) H.
- the anti-human GPC3 antibody H chain gene NTA2L or NTA2R was produced by linking LALA-S3K-G1d-CD3 or LALA-G3S3E-G1d downstream of GC33 (2) H.
- NTA1L, NTA1R, NTA2L, NTA2R (H chain) and GC33-k0 (L chain) genes were incorporated into animal cell expression vectors to prepare expression vectors for the genes. According to the combinations shown below, these vectors were introduced into FreeStyle293 cells (Invitrogen) by methods known to those skilled in the art, so that each polypeptide assembly shown below was transiently expressed. As shown below, the name of the polypeptide aggregate is indicated by indicating the combination of the introduced genes in the order of the first H chain / second H chain / L chain. NTA1L / NTA1R / GC33-k0 NTA2L / NTA2R / GC33-k0
- CM culture supernatant
- the rProtein A Sepharose Fast Flow column (GE Healthcare) equilibrated with D-PBS was loaded with CM filtered through a ⁇ 0.22 ⁇ m filter, and washing 1, 2, and elution 1 steps were performed using the buffers shown in Table 1. It was. The amount of CM loaded was adjusted so that the amount of antibody loaded was 20 mg / mL resin. The components contained in the eluted fraction were identified by size exclusion chromatography analysis of the collected eluted fraction.
- NTA2R / GC33-k0 Homoantibodies to GPC3 (NTA2R / GC33-k0) were detected in 76% of CMs expressing NTA1L / NTA1R / GC33-k0, whereas NTA2L / NTA2R / GC33-k0 was expressed. Only about 2% was detected in commercials. In other words, in addition to the mutation that replaces the EU numbering 435th His with Arg, the EU numbering 356th Asp in the polypeptide sequence of one H chain is used to efficiently form a heteromolecule of each H chain.
- the target GPC3 ERY10- is obtained only by the purification step using protein A. It was revealed that hetero-associated polypeptide aggregates having the same molecular form as 1 can be efficiently purified with a purity of 98% or more.
- ERY17-2_Hh (SEQ ID NO: 73, the amino terminal 19 amino acids as a signal sequence is a mature sequence by a method known to those skilled in the art such as PCR using a primer added with an appropriate sequence similar to the method described above.
- ERY17-2_L (SEQ ID NO: 74, amino terminal 19 amino acid as signal sequence is not included in mature sequence)
- ERY17-3_Hh (SEQ ID NO: 75, amino terminal 19 as signal sequence)
- a series of expressions in which polynucleotides encoding ERY17-3_L (SEQ ID NO: 76, amino acid terminal 19 amino acid as a signal sequence is not included in the mature sequence) are inserted. A vector was created.
- GPC3 ERY17-2 Polypeptides encoded by the polynucleotide inserted into the expression vector: GPC3 ERY8-2_Hk, GPC3 ERY7_L, ERY17-2_Hh (SEQ ID NO: 73, the amino terminal 19 amino acids which are signal sequences are not included in the mature sequence), ERY17-2_L (SEQ ID NO: 74, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequence)
- Target molecule GPC3 ERY17-3 Polypeptides encoded by the polynucleotide inserted in the expression vector: GPC3 ERY8-2_Hk, GPC3 ERY7_L, ERY17-3_Hh (SEQ ID NO: 75, the amino terminal 19 amino acids which are signal sequences are not included in the mature sequence), ERY17-3_L (SEQ ID NO: 76, signal sequence amino terminal 19 amino acids are not included in the mature sequence)
- mice Human lung squamous cell carcinoma cell line PC-10 (Immunobiology Laboratories) 1 ⁇ 10 7 cells and Matrigel basement membrane matrix (BD) are mixed, and NOD scid mice (CLEA Japan, female, 7W) Transplanted subcutaneously.
- the day of transplantation was defined as day 0.
- the mice On the day before transplantation and on days 13, 17, 21, and 25, the mice were intraperitoneally administered with 0.2 mg / animal of anti-asialo GM1 antibody (Wako Pure Chemical Industries).
- grouping was performed according to the tumor size and body weight, and on the 14th day after transplantation, T cells obtained by the expansion culture were transplanted into the peritoneal cavity at 3 ⁇ 10 7 cells / mouse.
- GPC ERY17-2 was intravenously administered at 30 ⁇ g / mouse.
- GPC ERY17-2 was administered five times on days 14, 15, 16, 17, and 18.
- GPC3 ERY17-2-M20 (FIG. 19A) in which the sequence of the binding domain of CD3 epsilon of GPC3 ERY17-2 was changed was produced. That is, an expression vector of CD3 antibody (M20) is used as a template, and ERY17-2-M20_Hh (by a method known to those skilled in the art such as PCR using a primer to which an appropriate sequence similar to the above method is added.
- GPC3 ERY17-2-M20 Expression vectors of GPC3 ERY8-2_Hk, GPC3 ERY7_L, ERY17-2-M20_Hh (SEQ ID NO: 77), and ERY17-2-M20_L (SEQ ID NO: 78) are all FreeStyle293. -Introduced into F cells and transiently expressed GPC3 ERY17-2-M20. The obtained culture supernatant was filtered through a ⁇ 0.22 ⁇ m filter and then loaded onto an equilibrated rProtein A Sepharose Fast Flow column (GE Healthcare). Purified GPC3 ERY17-2-M20 was obtained by carrying out the steps of washing 1, 2 and elution 1 with the buffers shown in Table 3.
- EpCAM ERY17-2 and EpCAM ERY17-3 (1) Preparation of EpCAM ERY17-2 and EpCAM ERY17-3 Next, molecules having the desired activity even if the target cancer antigen changes The creation of was attempted.
- EpCAM ERY17-2 (FIG. 19A) in which Fab for GPC3 of GPC3 ERY17-2 was changed to Fab for EpCAM
- EpCAM ERY17-3 (FIG. 19B) in which Fab for GPC3 of GPC3 ERY17-3 was changed to Fab for EpCAM, respectively. Made.
- EpCAM ERY17_Hk (SEQ ID NO: 79, signal) is used by a method known to those skilled in the art such as a PCR method using an EpCAM antibody expression vector as a template and a primer added with an appropriate sequence similar to the method described above.
- the amino terminal 19 amino acids that are sequences are not included in the mature sequence
- polynucleotides that encode EpCAM ERY17_L (SEQ ID NO: 80, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequences) are inserted.
- a series of expressed vectors was produced.
- EpCAM ERY17-2 Polypeptide encoded by polynucleotide inserted into expression vector: EpCAM ERY17_Hk (SEQ ID NO: 79, amino terminal 19 amino acids as signal sequence is not included in mature sequence), EpCAM ERY17_L (SEQ ID NO: 80, signal The amino terminal 19 amino acids that are sequences are not included in the mature sequence), ERY17-2_Hh, ERY17-2_L
- EpCAM ERY17-3 Polypeptides encoded by polynucleotides inserted into expression vectors: EpCAM ERY17_Hk, EpCAM ERY17_L, ERY17-3_Hh, ERY17-3_L
- M12_TH2h (SEQ ID NO: 85) in which several amino acids of CH1 of M12 H chain (SEQ ID NO: 81) are substituted with Lys, and several amino acids of CL in L chain (SEQ ID NO: 82) are Glu.
- Expression vectors into which polynucleotides encoding each substituted M12_TL17 (SEQ ID NO: 86) were inserted were prepared by methods known to those skilled in the art.
- GC33 (2) _TH13k (SEQ ID NO: 87)
- GC33 (2) _TH15k (SEQ ID NO: 88) in which several amino acids of CH1 of the H chain (SEQ ID NO: 83) of GC33 (2) are substituted with Glu
- the polynucleotides encoding GC33 (2) _TL16 (SEQ ID NO: 89) and GC33 (2) _TL19 (SEQ ID NO: 90), in which several amino acids of CL in the L chain (SEQ ID NO: 84) are substituted with Lys
- the inserted expression vector was prepared by methods known to those skilled in the art.
- a combination of expression vectors encoding the sequences shown below was introduced into FreeStyle293-F cells to transiently express each target molecule.
- T.A. Target molecule GM1 Expression vectors: M12_TH2h (SEQ ID NO: 85), M12_TL17 (SEQ ID NO: 86), GC33 (2) _TH13k (SEQ ID NO: 87), and GC33 (2) _TL16 (SEQ ID NO: 89)
- Target molecule GM2 Expression vectors: M12_TH2h (SEQ ID NO: 85), M12_TL17 (SEQ ID NO: 86), GC33 (2) _TH15k (SEQ ID NO: 88), and GC33 (2) _TL19 (SEQ ID NO: 90)
- Target molecule GM0 Expression vector: M12 H chain (SEQ ID NO: 81), M12 L chain (SEQ ID NO: 82), GC33 (2) H chain (SEQ ID NO: 83), and GC33 (2) L chain (SEQ ID NO: : 84)
- the antibody was purified from the obtained culture supernatant by a method known to those skilled in the art using rProtein A Sepharose TM Fast Flow (GE Healthcare).
- an EGFR antibody expression vector is used as a template, and EGFR ERY17_Hk (SEQ ID NO: 91, signal The amino terminal 19 amino acids that are sequences are not included in the mature sequence), and polynucleotides encoding EGFR ERY17_L (SEQ ID NO: 92, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequences) are inserted.
- EGFR ERY17_Hk SEQ ID NO: 91, signal The amino terminal 19 amino acids that are sequences are not included in the mature sequence
- polynucleotides encoding EGFR ERY17_L SEQ ID NO: 92, the amino terminal 19 amino acids that are signal sequences are not included in the mature sequences
- Target molecule EGFR ERY17-2 Polypeptide encoded by the polynucleotide inserted in the expression vector: EGFR ERY17_Hk (SEQ ID NO: 91, the amino terminal 19 amino acids as the signal sequence is not included in the mature sequence), EGFR ERY17_L (SEQ ID NO: 92, signal The amino terminal 19 amino acids that are sequences are not included in the mature sequence), ERY17-2_Hh, ERY17-2_L
- a novel polypeptide group that maintains the strong antitumor activity of BiTE and the excellent safety property that does not induce cytokine storm in a cancer antigen-independent manner and has a long blood half-life. Coalition was provided.
- the cytotoxicity-inducing therapeutic agent containing the polypeptide aggregate brings about cytotoxicity targeting various cells including cancer cells, Cancer can be treated or prevented.
- the patient not only is the safety high, but also a desirable treatment that is less burdensome and highly convenient can be performed.
Abstract
Description
〔1〕 下記のドメイン;
(1)抗原結合ドメイン、
(2)Fcγ受容体に対する結合活性が低下しているFc領域を含むドメイン、及び、
(3)T細胞受容体複合体結合ドメイン、
を含むポリペプチド会合体。
〔2〕 T細胞受容体複合体結合ドメインがT細胞受容体結合ドメインである、〔1〕に記載のポリペプチド会合体。
〔3〕 T細胞受容体複合体結合ドメインがCD3結合ドメインである、〔1〕に記載のポリペプチド会合体。
〔4〕 抗原結合ドメインが二価の抗原結合ドメインである、〔1〕から〔3〕のいずれかに記載のポリペプチド会合体。
〔5〕 二価の抗原結合ドメインがF(ab’)2の構造を有するドメインである、〔4〕に記載のポリペプチド会合体。
〔6〕 F(ab’)2の構造を有するドメインの重鎖定常領域を構成する二つのポリペプチドがFc領域を構成する二つのポリペプチドの各々に連結された、〔5〕に記載のポリペプチド会合体。
〔7〕 CD3結合ドメインがFc領域を構成する一つ又は二つのCH3に連結された、〔6〕に記載のポリペプチド会合体。
〔8〕 CD3結合ドメインを構成する重鎖Fv断片がFc領域を構成する一方のCH3に連結され、CD3結合ドメインを構成する軽鎖Fv断片がFc領域を構成するもう一方のCH3に連結された、〔7〕に記載のポリペプチド会合体。
〔9〕 CD3結合ドメインを構成する重鎖Fv断片に抗体のCH1ドメイン、及び、軽鎖Fv断片に抗体のCLドメインが連結された、〔8〕に記載のポリペプチド会合体。
〔10〕 CD3結合ドメインがF(ab’)2を構成する一つ又は二つのCLに連結された、〔6〕に記載のポリペプチド会合体。
〔11〕 CD3結合ドメインがF(ab’)2を構成する一つ又は二つのVHに連結された、〔6〕に記載のポリペプチド会合体。
〔12〕 CD3結合ドメインがF(ab’)2を構成する一つ又は二つのVLに連結された、〔6〕に記載のポリペプチド会合体。
〔13〕 CD3結合ドメインがFvである、〔1〕から〔12〕のいずれかに記載のポリペプチド会合体。
〔14〕 CD3結合ドメインがFabである、〔1〕から〔7〕及び〔10〕から〔12〕のいずれかに記載のポリペプチド会合体。
〔15〕 CD3結合ドメインがscFvである、〔1〕から〔7〕及び〔10〕から〔12〕のいずれかに記載のポリペプチド会合体。
〔16〕 CD3結合ドメインが一価である、〔1〕から〔15〕のいずれかに記載のポリペプチド会合体。
〔17〕 抗原結合ドメインが一価のscFv及び一価のFabである、〔1〕から〔3〕のいずれかに記載のポリペプチド会合体。
〔18〕 一価のscFvがCD3結合ドメインを構成するscFvを介してFc領域を構成する一つのポリペプチドに、一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一つのポリペプチドに各々連結され、当該Fabの軽鎖Fv断片がCL領域と連結された、〔17〕に記載のポリペプチド会合体。
〔19〕 抗原結合ドメインが二価のscFvである、〔1〕から〔3〕のいずれかに記載のポリペプチド会合体。
〔20〕 一価のscFvがCD3結合ドメインを構成する重鎖Fv断片を介してFc領域を構成する一つのポリペプチドに、他方の一価のscFvがCD3結合ドメインを構成する軽鎖Fv断片を介してFc領域を構成する他方の一つのポリペプチドに連結された、〔19〕に記載のポリペプチド会合体。
〔21〕 一価のscFvがCD3結合ドメインを構成するscFvを介してFc領域を構成する一つのポリペプチドに、他方の一価のscFvがFc領域を構成する他方の一つのポリペプチドに連結された、〔19〕に記載のポリペプチド会合体。
〔22〕 抗原結合ドメイン、及び、T細胞受容体複合体結合ドメインが各々一価のFabである、〔1〕から〔3〕のいずれかに記載のポリペプチド会合体。
〔23〕 抗原結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、T細胞受容体結合ドメインを構成するFabの重鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結された、〔22〕に記載のポリペプチド会合体。
〔24〕 抗原結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、T細胞受容体結合ドメインを構成するFabの軽鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの重鎖Fv断片がCL領域と連結された、〔22〕に記載のポリペプチド会合体。
〔25〕 抗原結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、T細胞受容体結合ドメインを構成するFabの重鎖Fv断片がCL領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCH1領域と連結された、〔22〕に記載のポリペプチド会合体。
〔26〕 T細胞受容体結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、抗原結合ドメインを構成するFabの軽鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの重鎖Fv断片がCL領域と連結された、〔22〕に記載のポリペプチド会合体。
〔27〕 T細胞受容体結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、抗原結合ドメインを構成するFabの重鎖Fv断片がCL領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCH1領域と連結された、〔22〕に記載のポリペプチド会合体。
〔28〕 (1)抗原に結合する一価のFab構造の重鎖Fv断片がCH1領域を介して前記Fc領域を構成する一方のポリペプチドに連結され、当該Fab構造の軽鎖Fv断片がCL領域と連結された抗原結合ドメイン、及び、
(2)T細胞受容体複合体に結合する一価のFab構造の重鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fab構造の軽鎖Fv断片がCL領域と連結されたT細胞受容体複合体結合ドメイン、
を含むポリペプチド会合体であって、抗原結合ドメイン中の重鎖Fv断片と抗原結合ドメイン中の軽鎖Fv断片またはT細胞受容体結合ドメイン中の重鎖Fv断片とT細胞受容体結合ドメイン中の軽鎖Fv断片が会合するようにCH1領域とCL領域の電荷が制御されている、〔22〕に記載のポリペプチド会合体。
〔29〕 T細胞受容体複合体結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基および抗原結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有する、〔28〕に記載のポリペプチド会合体。
〔30〕 抗原結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基およびT細胞受容体複合体結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有する、〔28〕に記載のポリペプチド会合体。
〔31〕 T細胞受容体複合体結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基および抗原結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有し、抗原結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基およびT細胞受容体複合体結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有する、〔28〕に記載のポリペプチド会合体。
〔32〕 T細胞受容体複合体結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基およびT細胞受容体結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに異種の電荷を有する、〔29〕又は〔31〕に記載のポリペプチド会合体。
〔33〕 抗原結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基および抗原結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基がともに異種の電荷を有する、〔30〕または〔31〕に記載のポリペプチド会合体。
〔34〕 T細胞受容体複合体結合ドメインがT細胞受容体結合ドメインである、〔22〕から〔33〕のいずれかに記載のポリペプチド会合体。
〔35〕 T細胞受容体結合ドメインがCD3結合ドメインである、〔34〕に記載のポリペプチド会合体。
〔36〕 CH1領域のアミノ酸残基およびCL領域のアミノ酸残基が、以下の(a)~(f)に示される1組又は2組以上のアミノ酸残基の組からなる群;
(a)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング180位のアミノ酸残基、
(b)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング131位のアミノ酸残基
(c)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング164位のアミノ酸残基
(d)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング138位のアミノ酸残基
(e)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング123位のアミノ酸残基
(f)CH1領域のアミノ酸残基であってEUナンバリング175位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング160位のアミノ酸残基
より選択され、CH1領域のアミノ酸残基とCL領域のアミノ酸残基とが互いに異種の電荷を有するアミノ酸残基である、〔32〕又は〔33〕のいずれかに記載のポリペプチド会合体。
〔37〕 さらに、以下の(g)に示されるアミノ酸残基の組を含む群より選択される、〔36〕に記載のポリペプチド会合体。
(g)CH1領域のアミノ酸残基であってEUナンバリング213位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング123位のアミノ酸残基
〔38〕 前記異種の電荷を有するアミノ酸残基が、以下の(X)または(Y)のいずれかの群;
(X)グルタミン酸(E)、アスパラギン酸(D);
(Y)リジン(K)、アルギニン(R)、ヒスチジン(H);
に含まれるアミノ酸残基から選択される、〔36〕又は〔37〕に記載のポリペプチド会合体。
〔39〕 前記異種の電荷を有するアミノ酸残基が、CH1領域のアミノ酸残基であってEUナンバリング175位のアミノ酸残基がLys、CL領域のアミノ酸残基であってEUナンバリング180位、131位及び160位のアミノ酸残基がいずれもGluである、〔36〕から〔38〕のいずれかに記載のポリペプチド会合体。
〔40〕 前記異種の電荷を有するアミノ酸残基が、CH1領域のアミノ酸残基であってEUナンバリング147位及び175位のアミノ酸残基がGlu、CL領域のアミノ酸残基であってEUナンバリング180位、131位及び160位のアミノ酸残基がいずれもLysである、〔36〕から〔38〕のいずれかに記載のポリペプチド会合体。
〔41〕 さらに、CH1領域のアミノ酸残基であってEUナンバリング213位のアミノ酸残基がGluであり、CL領域のアミノ酸残基であってEUナンバリング123位のアミノ酸残基がLysである、〔40〕に記載のポリペプチド会合体。
〔42〕 Fc領域がFcγI、FcγIIA、FcγIIB、FcγIIIA及び/又はFcγIIIBのいずれかのFcγ受容体に対する結合活性が低下しているFc領域である、〔1〕から〔41〕のいずれかに記載のポリペプチド会合体。
〔43〕 Fc領域が、配列番号:23に記載のFc領域、配列番号:24に記載のFc領域、配列番号:25に記載のFc領域、又は配列番号:26に記載のFc領域を構成するアミノ酸が変異しているFc領域であることを特徴とする、〔1〕から〔42〕のいずれかに記載のポリペプチド会合体。
〔44〕 Fc領域を構成するアミノ酸のうちEUナンバリングに従って特定される下記のいずれかのアミノ酸;
118位から260位のアミノ酸配列が配列番号:24に記載の配列、261位から447位のアミノ酸配列が配列番号:26に記載の配列であるFc領域である、〔43〕に記載のポリペプチド会合体。
〔45〕 Fc領域を構成するアミノ酸のうちEUナンバリングに従って特定される下記のいずれかのアミノ酸;
220位、226位、229位、231位、232位、233位、234位、235位、236位、237位、238位、239位、240位、264位、265位、266位、267位、269位、270位、295位、296位、297位、298位、299位、300位、325位、327位、328位、329位、330位、331位、332位、
が変異しているFc領域である、〔43〕に記載のポリペプチド会合体。
〔46〕 Fc領域が配列番号:23に記載のFc領域を構成するアミノ酸が変異しているFc領域であることを特徴とする、〔45〕に記載のポリペプチド会合体。
〔47〕 Fc領域を構成するアミノ酸のうちEUナンバリングに従って特定される下記のいずれかのアミノ酸;
233位、234位、235位、236位、237位、327位、330位、331位、
が対応するIgG2またはIgG4においてそのEUナンバリングが対応するアミノ酸に置換されたFc領域である、〔46〕に記載のポリペプチド会合体。
〔48〕 Fc領域を構成するアミノ酸のうちEUナンバリングに従って特定される下記のいずれかのアミノ酸;
234位、235位、297位、
が変異しているFc領域であることを特徴とする、〔46〕に記載のポリペプチド会合体。
〔49〕 234位のアミノ酸がアラニン、235位のアミノ酸がアラニン、及び/又は、297位のアミノ酸がアラニンに変異していることを特徴とする、〔48〕に記載のポリペプチド会合体。
〔50〕 Fc領域を構成する二つのポリペプチドの配列が互いに異なる配列を有することを特徴とする、〔43〕から〔49〕のいずれかに記載のポリペプチド会合体。
〔51〕 Fc領域を構成する二つのポリペプチドの一方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される349位のアミノ酸がシステイン、366位のアミノ酸がトリプトファンに、他方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される356位のアミノ酸がシステイン、366位のアミノ酸がセリンに、368位のアミノ酸がアラニンに、407位のアミノ酸がバリンに変異していることを特徴とする、〔1〕から〔50〕のいずれかに記載のポリペプチド会合体。
〔52〕 Fc領域を構成する二つのポリペプチドの一方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される356位のアミノ酸がリジンに、他方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される439位のアミノ酸がグルタミン酸に変異し、いずれか一方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される435位のアミノ酸がアルギニンに変異していることを特徴とする、〔1〕から〔50〕のいずれかに記載のポリペプチド会合体。
〔53〕 Fc領域を構成する二つのポリペプチドのカルボキシ末端に存在する配列GKが欠失していることを特徴とする、〔51〕又は〔52〕に記載のポリペプチド会合体。
〔54〕 抗原結合ドメインが同一のエピトープに結合する、〔1〕から〔53〕のいずれかに記載のポリペプチド会合体。
〔55〕 同一のエピトープが配列番号:2に記載のアミノ酸配列からなるタンパク質中に存在する、〔54〕に記載のポリペプチド会合体。
〔56〕 同一のエピトープが配列番号:4に記載のアミノ酸配列からなるタンパク質中に存在する、〔54〕に記載のポリペプチド会合体。
〔57〕 抗原結合ドメインが互いに異なるエピトープに結合する、〔1〕から〔53〕のいずれかに記載のポリペプチド会合体。
〔58〕 異なるエピトープが配列番号:2に記載のアミノ酸配列からなるタンパク質中に存在する、〔57〕に記載のポリペプチド会合体。
〔59〕 異なるエピトープが配列番号:4に記載のアミノ酸配列からなるタンパク質中に存在する、〔57〕に記載のポリペプチド会合体。
〔60〕 〔1〕から〔59〕のいずれかに記載のポリペプチド会合体をコードするポリヌクレオチド。
〔61〕 〔60〕に記載のポリヌクレオチドを含むベクター。
〔62〕 〔61〕に記載のベクターを保持する細胞。
〔63〕 〔62〕に記載の細胞を培養し培養上清からポリペプチド会合体を回収することを含むポリペプチド会合体の製造方法。
〔64〕 〔1〕から〔59〕のいずれかに記載のポリペプチド会合体を有効成分として含む細胞傷害誘導治療剤。
〔65〕 細胞傷害誘導治療剤が癌治療剤である、〔64〕に記載の治療剤。
〔66〕 癌が肝癌又は肺癌である、〔65〕に記載の治療剤。
〔67〕 〔1〕から〔59〕のいずれかに記載のポリペプチド会合体を治療が必要な患者に投与することを特徴とする、癌の治療又は予防方法。
〔68〕 癌が肝癌又は肺癌である、〔67〕に記載の治療又は予防方法。
本明細書において、抗体とは、天然のものであるかまたは部分的もしくは完全合成により製造された免疫グロブリンをいう。抗体はそれが天然に存在する血漿や血清等の天然資源や抗体を産生するハイブリドーマ細胞の培養上清から単離され得るし、または遺伝子組換え等の手法を用いることによって部分的にもしくは完全に合成され得る。抗体の例としては免疫グロブリンのアイソタイプおよびそれらのアイソタイプのサブクラスが好適に挙げられる。ヒトの免疫グロブリンとして、IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgD、IgE、IgMの9種類のクラス(アイソタイプ)が知られている。本発明の抗体には、これらのアイソタイプのうちIgG1、IgG2、IgG3、IgG4が含まれ得る。
-GPC3のような膜蛋白質の構造を維持して免疫刺激が与えられ得る
-免疫抗原を精製する必要が無い
より具体的には、例えば細胞融合促進剤の存在下で通常の栄養培養液中で、前記細胞融合が実施され得る。融合促進剤としては、例えばポリエチレングリコール(PEG)、センダイウイルス(HVJ)等が使用され、更に融合効率を高めるために所望によりジメチルスルホキシド等の補助剤が添加されて使用される。
-グアニジン超遠心法(Biochemistry (1979) 18 (24), 5294-5299)
-AGPC法(Anal. Biochem. (1987) 162 (1), 156-159)
(1)ハイブリドーマから得られたcDNAによってコードされるV領域を含む抗体をGPC3発現細胞に接触させる工程、
(2)GPC3発現細胞と抗体との結合を検出する工程、および
(3)GPC3発現細胞に結合する抗体を選択する工程。
(1)哺乳類細胞、:CHO、COS、ミエローマ、BHK(baby hamster kidney)、Hela、Veroなど
(2)両生類細胞:アフリカツメガエル卵母細胞など
(3)昆虫細胞:sf9、sf21、Tn5など
-酵母:サッカロミセス・セレビシエ(Saccharomyces serevisiae)などのサッカロミセス(Saccharomyces)属、メタノール資化酵母(Pichia pastoris)などのPichia属
-糸状菌:アスペスギルス・ニガー(Aspergillus niger)などのアスペルギルス(Aspergillus)属
本明細書において「抗原結合ドメイン」とは、抗原の一部または全部に特異的に結合し且つ相補的である領域を含んで成る抗体の部分をいう。抗原の分子量が大きい場合、抗体は抗原の特定部分にのみ結合することができる。当該特定部分はエピトープと呼ばれる。抗原結合ドメインは一または複数の抗体の可変ドメインより提供され得る。好ましくは、抗原結合ドメインは抗体軽鎖可変領域(VL)と抗体重鎖可変領域(VH)とを含む。こうした抗原結合ドメインの例としては、「scFv(single chain Fv)」、「単鎖抗体(single chain antibody)」、「Fv」、「scFv2(single chain Fv 2)」、「Fab」または「F(ab')2」等が好適に挙げられる。
特異的とは、特異的に結合する分子の一方の分子がその一または複数の結合する相手方の分子以外の分子に対しては何ら有意な結合を示さない状態をいう。また、抗原結合ドメインが、ある抗原中に含まれる複数のエピトープのうち特定のエピトープに対して特異的である場合にも用いられる。また、抗原結合ドメインが結合するエピトープが複数の異なる抗原に含まれる場合には、当該抗原結合ドメインを有するポリペプチド会合体は当該エピトープを含む様々な抗原と結合することができる。
本明細書において抗原は特に限定されず、CD3を除きどのような抗原でもよい。抗原の例としては、例えば、受容体、癌抗原、MHC抗原、分化抗原等が好適に挙げられる。受容体の例としては、例えば、造血因子受容体ファミリー、サイトカイン受容体ファミリー、チロシンキナーゼ型受容体ファミリー、セリン/スレオニンキナーゼ型受容体ファミリー、TNF受容体ファミリー、Gタンパク質共役型受容体ファミリー、GPIアンカー型受容体ファミリー、チロシンホスファターゼ型受容体ファミリー、接着因子ファミリー、ホルモン受容体ファミリー、等の受容体ファミリーに属する受容体などを挙げることができる。これら受容体ファミリーに属する受容体、及びその特徴に関しては、多数の文献、例えば、Cooke BA., King RJB., van der Molen HJ. ed. New Comprehesive Biochemistry Vol.18B "Hormones and their Actions Part II"pp.1-46 (1988) Elsevier Science Publishers BV.、又は、宮坂昌之監修, 細胞工学別冊ハンドブックシリーズ「接着因子ハンドブック」(1994) (秀潤社, 東京, 日本)等のレビューの他、Patthy(Cell (1990) 61 (1), 13-14)、Ullrichら(Cell (1990) 61 (2), 203-212)、Massague(eにはアキュート・アクセント記号が付く)(Cell (1992) 69 (6), 1067-1070)、Miyajimaら(Annu. Rev. Immunol. (1992) 10, 295-331)、Tagaら(FASEB J. (1992) 6, 3387-3396)、Fantlら(Annu. Rev. Biochem. (1993), 62, 453-481)、Smithら(Cell (1994) 76 (6) 959-962)、Flower DR. Biochim. Biophys. Acta, Flower(Biochim. Biophys. Acta (1999) 1422 (3) 207-234等に記載されている。
抗原中に存在する抗原決定基を意味するエピトープは、本明細書において開示されるポリペプチド会合体中の抗原結合ドメインが結合する抗原上の部位を意味する。よって、例えば、エピトープは、その構造によって定義され得る。また、当該エピトープを認識するポリペプチド会合体中の抗原に対する結合活性によっても当該エピトープが定義され得る。抗原がペプチド又はポリペプチドである場合には、エピトープを構成するアミノ酸残基によってエピトープを特定することも可能である。また、エピトープが糖鎖である場合には、特定の糖鎖構造によってエピトープを特定することも可能である。
FACSCantoTM II
FACSAriaTM
FACSArrayTM
FACSVantageTM SE
FACSCaliburTM (いずれもBD Biosciences社の商品名)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
Cell Lab Quanta / Cell Lab Quanta SC(いずれもBeckman Coulter社の商品名)
本明細書において、「Fv(variable fragment)」という用語は、抗体の軽鎖可変領域(VL(light chain variable region))と抗体の重鎖可変領域(VH(heavy chain variable region))とのペアからなる抗体由来の抗原結合ドメインの最小単位を意味する。1988年にSkerraとPluckthunは、バクテリアのシグナル配列の下流に抗体の遺伝子を挿入し大腸菌中で当該遺伝子の発現を誘導することによって、均一でかつ活性を保持した状態で大腸菌のペリプラズム画分から調製されることを見出した(Science (1988) 240 (4855), 1038-1041)。ペリプラズム画分から調製されたFvは、抗原に対する結合を有する態様でVHとVLが会合していた。
二価のscFvのうち一価のscFvがCD3結合ドメインを構成する重鎖Fv断片を介してFc領域を構成する一つのポリペプチドに、他方の一価のscFvがCD3結合ドメインを構成する軽鎖Fv断片を介してFc領域を構成する他方の一つのポリペプチドに連結された二価の抗原結合ドメインが二価のscFvである(1)二価の抗原結合ドメイン、(2)IgG1、IgG2a、IgG3又はIgG4のFc領域を構成するアミノ酸のうちFcγ受容体に対する結合活性を有しないFc領域を含むドメイン、及び、(3)少なくとも一価のCD3結合ドメイン、
を含むポリペプチド会合体等において軽鎖Fv断片及び重鎖Fv断片が、抗原であるCD3に対する結合を有する態様で会合しCD3結合ドメインを構成する一組のFvも好適に含まれる。
本明細書において、「scFv」、「単鎖抗体」、または「sc(Fv)2」という用語は、単一のポリペプチド鎖内に、重鎖および軽鎖の両方に由来する可変領域を含むが、定常領域を欠いている抗体断片を意味する。一般に、単鎖抗体は、抗原結合を可能にすると思われる所望の構造を形成するのを可能にする、VHドメインとVLドメインの間のポリペプチドリンカーをさらに含む。単鎖抗体は、The Pharmacology of Monoclonal Antibodies, 113巻, Rosenburg、及び、Moore編, Springer-Verlag, New York, 269~315(1994)においてPluckthunによって詳細に考察されている。同様に、国際特許出願公開WO1988/001649および米国特許第4,946,778号および同第5,260,203号を参照。特定の態様において、単鎖抗体はまた、二重特異性であるか、かつ/またはヒト化され得る。
[VL]リンカー[VH]リンカー[VH]リンカー[VL]
[VH]リンカー[VL]リンカー[VL]リンカー[VH]
[VH]リンカー[VH]リンカー[VL]リンカー[VL]
[VL]リンカー[VL]リンカー[VH]リンカー[VH]
[VL]リンカー[VH]リンカー[VL]リンカー[VH]
Ser
Gly・Ser
Gly・Gly・Ser
Ser・Gly・Gly
Gly・Gly・Gly・Ser(配列番号:5)
Ser・Gly・Gly・Gly(配列番号:6)
Gly・Gly・Gly・Gly・Ser(配列番号:7)
Ser・Gly・Gly・Gly・Gly(配列番号:8)
Gly・Gly・Gly・Gly・Gly・Ser(配列番号:9)
Ser・Gly・Gly・Gly・Gly・Gly(配列番号:10)
Gly・Gly・Gly・Gly・Gly・Gly・Ser(配列番号:11)
Ser・Gly・Gly・Gly・Gly・Gly・Gly(配列番号:12)
(Gly・Gly・Gly・Gly・Ser(配列番号:7))n
(Ser・Gly・Gly・Gly・Gly(配列番号:8))n
[nは1以上の整数である]等を挙げることができる。但し、ペプチドリンカーの長さや配列は目的に応じて当業者が適宜選択することができる。
「Fab」は、一本の軽鎖、ならびに一本の重鎖のCH1領域および可変領域から構成される。Fab分子の重鎖は、別の重鎖分子とのジスルフィド結合を形成できない。
本明細書において開示されるポリペプチド会合体を構成するFc領域はモノクローナル抗体等の抗体をペプシン等の蛋白質分解酵素にて部分消化した後に、断片をプロテインAカラム、あるいはプロテインGカラムに吸着させた後に、適切な溶出バッファー等により溶出させることにより好適に取得され得る。かかる蛋白質分解酵素としてはpH等の酵素の反応条件を適切に設定することによりモノクローナル抗体等の抗体を消化し得るものであれば特段の限定はされず、例えば、ペプシンやフィシン等が例示できる。
Fcγ受容体とは、IgG1、IgG2、IgG3、IgG4モノクローナル抗体のFc領域に結合し得る受容体をいい、実質的にFcγ受容体遺伝子にコードされるタンパク質のファミリーのいかなるメンバーをも意味する。ヒトでは、このファミリーには、アイソフォームFcγRIa、FcγRIbおよびFcγRIcを含むFcγRI(CD64);アイソフォームFcγRIIa(アロタイプH131およびR131を含む)、FcγRIIb(FcγRIIb-1およびFcγRIIb-2を含む)およびFcγRIIcを含むFcγRII(CD32);およびアイソフォームFcγRIIIa(アロタイプV158およびF158を含む)およびFcγRIIIb(アロタイプFcγRIIIb-NA1およびFcγRIIIb-NA2を含む)を含むFcγRIII(CD16)、並びにいかなる未発見のヒトFcγR類またはFcγRアイソフォームまたはアロタイプも含まれるが、これらに限定されるものではない。FcγRは、ヒト、マウス、ラット、ウサギおよびサルを含むが、これらに限定されるものではない、いかなる生物由来でもよい。マウスFcγR類には、FcγRI(CD64)、FcγRII(CD32)、FcγRIII(CD16)およびFcγRIII-2(CD16-2)、並びにいかなる未発見のマウスFcγR類またはFcγRアイソフォームまたはアロタイプも含まれるが、これらに限定されない。こうしたFcγ受容体の好適な例としてはヒトFcγI(CD64)、FcγIIA(CD32)、FcγIIB(CD32)、FcγIIIA(CD16)及び/又はFcγIIIB(CD16)が挙げられる。FcγIのポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:13(NM_000566.3)及び14(NP_000557.1)に、FcγIIAのポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:15(BC020823.1)及び16(AAH20823.1)に、FcγIIBのポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:17(BC146678.1)及び18(AAI46679.1)に、FcγIIIAのポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:19(BC033678.1)及び20(AAH33678.1)に、及びFcγIIIBのポリヌクレオチド配列及びアミノ酸配列は、それぞれ配列番号:21(BC128562.1)及び22(AAI28563.1)に記載されている(カッコ内はRefSeq登録番号を示す)。Fcγ受容体が、IgG1、IgG2、IgG3、IgG4モノクローナル抗体のFc領域に結合活性を有するか否かは、上記に記載されるFACSやELISAフォーマットのほか、ALPHAスクリーン(Amplified Luminescent Proximity Homogeneous Assay)や表面プラズモン共鳴(SPR)現象を利用したBIACORE法等によって確認され得る(Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010)。
Fc領域がFcγI、FcγIIA、FcγIIB、FcγIIIA及び/又はFcγIIIBのいずれかのFcγ受容体に対する結合活性が低下していることは、上記に記載されるFACSやELISAフォーマットのほか、ALPHAスクリーン(Amplified Luminescent Proximity Homogeneous Assay)や表面プラズモン共鳴(SPR)現象を利用したBIACORE法等によって確認することができる(Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010)。
(a)L234F、L235E、P331S、
(b)C226S、C229S、P238S、
(c)C226S、C229S、
(d)C226S、C229S、E233P、L234V、L235A
が施されているFc領域、又は、231位から238位のアミノ酸配列が欠失したFc領域を有するポリペプチド会合体も適宜使用され得る。
(e)H268Q、V309L、A330S、P331S
(f)V234A
(g)G237A
(h)V234A、G237A
(i)A235E、G237A
(j)V234A、A235E、G237A
が施されているFc領域を有するポリペプチド会合体も適宜使用され得る。
(k)F241A
(l)D265A
(m)V264A
が施されているFc領域を有するポリペプチド会合体も適宜使用され得る。
(n)L235A、G237A、E318A
(o)L235E
(p)F234A、L235A
が施されているFc領域を有するポリペプチド会合体も適宜使用され得る。
本明細書において、Fcγ受容体に対する結合活性が低下しているFc領域としては、二重特異性抗体(bispecific抗体)を起源とするFc領域も適宜使用される。二重特異性抗体とは、二つの異なる特異性を有する抗体である。IgG型の二重特異性抗体はIgG抗体を産生するハイブリドーマ二種を融合することによって生じるhybrid hybridoma(quadroma)によって分泌させることが出来る(Milstein C et al.Nature (1983) 305, 537-540)。
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される409位のアミノ酸がアスパラギン酸、370位のアミノ酸がグルタミン酸であり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される399位のアミノ酸がリジン、357位のアミノ酸がリジンであることを特徴とする、二つのポリペプチド(本態様では、370位のグルタミン酸に代えてアスパラギン酸であってもよく、370位のグルタミン酸に代えて392位のアスパラギン酸であってもよい)、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される409位のアミノ酸がアスパラギン酸、439位のアミノ酸がグルタミン酸であり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される399位のアミノ酸がリジン、356位のアミノ酸がリジンであることを特徴とする二つのポリペプチド(本態様では、439位のグルタミン酸に代えて360位のアスパラギン酸、392位のアスパラギン酸又は439位のアスパラギン酸であってもよい)、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される370位のアミノ酸がグルタミン酸、439位のアミノ酸がグルタミン酸であり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される357位のアミノ酸がリジン、356位のアミノ酸がリジンであることを特徴とする二つのポリペプチド、または、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される409位のアミノ酸がアスパラギン酸、370位のアミノ酸がグルタミン酸、439位のアミノ酸がグルタミン酸であり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される399位のアミノ酸がリジン、357位のアミノ酸がリジン、356位のアミノ酸がリジンであることを特徴とする二つのポリペプチド(本態様では、370位のアミノ酸をグルタミン酸に置換しなくてもよく、更に、370位のアミノ酸をグルタミン酸に置換しない上で、439位のグルタミン酸に代えてアスパラギン酸又は439位のグルタミン酸に代えて392位のアスパラギン酸であってもよい)、
が好適に用いられる。
本明細書において、Fcγ受容体に対する結合活性が低下しているFc領域として、上記の特徴に加えてFc領域のC末端のヘテロジェニティーが改善されたFc領域が適宜使用され得る。より具体的には、IgG1、IgG2、IgG3又はIgG4を起源とするFc領域を構成する二つのポリペプチドのアミノ酸配列のうちEUナンバリングに従って特定される446位のグリシン、及び447位のリジンが欠失したFc領域が提供される。
本明細書において、「T細胞受容体複合体結合ドメイン」とは、T細胞受容体複合体の一部または全部に特異的に結合し且つ相補的である領域を含んで成るT細胞受容体複合体抗体の部分をいう。T細胞受容体複合体は、T細胞受容体自身でもよいし、T細胞受容体とともにT細胞受容体複合体を構成するアダプター分子でもよい。アダプターとして好適なものはCD3である。
本明細書において、「T細胞受容体結合ドメイン」とは、T細胞受容体の一部または全部に特異的に結合し且つ相補的である領域を含んでなるT細胞受容体抗体の部分をいう。
本明細書において「CD3結合ドメイン」とは、CD3の一部または全部に特異的に結合し且つ相補的である領域を含んで成るCD3抗体の部分をいう。CD3結合ドメインは一または複数の抗体の可変ドメインより提供され得る。好ましくは、CD3結合ドメインはCD3抗体の軽鎖可変領域(VL)とCD3抗体の重鎖可変領域(VH)とを含む。こうしたCD3結合ドメインの例としては、「scFv(single chain Fv)」、「単鎖抗体(single chain antibody)」、「Fv」、「scFv2(single chain Fv 2)」、「Fab」または「F(ab')2」等が好適に挙げられる。
本発明に係るポリペプチド会合体は前記の、
(1)抗原結合ドメイン、
(2)Fcγ受容体に対する結合活性が低下しているFc領域を含むドメイン、及び、
(3)T細胞受容体複合体結合ドメイン、
を含むものであればよく、その構造は限定されない。本発明においては、T細胞受容体複合体結合ドメインは、好ましくはT細胞受容体結合ドメインあるいはCD3結合ドメインである。上記の各ドメインはペプチド結合で直接連結することができる。例えば、(1)抗原結合ドメインとしてF(ab')2を用い、(2)Fcγ受容体に対する結合活性が低下しているFc領域を含むドメインとしてこれらのFc領域を用いた場合に(1)に記載された抗原結合ドメインと(2)に記載されたFc領域を含むドメインとをペプチド結合で連結したときは、連結されたポリペプチドは抗体の構造を形成する。そのような抗体を作製するためには前述のハイブリドーマの培養液から精製する他、当該抗体を構成するポリペプチドをコードするポリヌクレオチドが安定に保持された所望の宿主細胞の培養液から当該抗体を精製することもできる。
(1)抗原に結合する一価のFab構造の重鎖Fv断片がCH1領域を介して前記Fc領域を構成する一方のポリペプチドに連結され、当該Fab構造の軽鎖Fv断片がCL領域と連結された抗原結合ドメイン、及び、
(2)T細胞受容体複合体に結合する一価のFab構造の重鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fab構造の軽鎖Fv断片がCL領域と連結されたT細胞受容体複合体結合ドメイン、
を含み、抗原結合ドメイン中の重鎖Fv断片と抗原結合ドメイン中の軽鎖Fv断片またはT細胞受容体結合ドメイン中の重鎖Fv断片とT細胞受容体結合ドメイン中の軽鎖Fv断片が会合するようにCH1領域とCL領域の電荷が制御されているポリペプチドが好適に挙げられる。本態様においては、抗原結合ドメイン中の重鎖Fv断片と抗原結合ドメイン中の軽鎖Fv断片またはT細胞受容体結合ドメイン中の重鎖Fv断片とT細胞受容体結合ドメイン中の軽鎖Fv断片が会合するようにCH1領域とCL領域の電荷が制御されていればよく、そのポリペプチド会合体の構造(会合制御構造)は特定の一構造に限定されない。
T細胞受容体結合ドメインの重鎖と軽鎖によりT細胞受容体結合ドメインのエピトープを認識し、また、抗原結合ドメインの重鎖と軽鎖により抗原のエピトープを認識するような二重特異性ポリペプチド会合体を取得したい場合、当該ポリペプチド会合体の生産に際して4種のそれぞれの鎖を発現させると理論上10種類のポリペプチド会合体分子が生産される可能性がある。
・CH1のEUナンバリング147位(例えば、配列番号:1に記載のアミノ酸配列における147位)のリジン(K)と、相対(接触)するCLのEUナンバリング180位のスレオニン(T)
・CH1のEUナンバリング147位のリジン(K)と、相対(接触)するCLのEUナンバリング131位のセリン(S)
・CH1のEUナンバリング147位のリジン(K)と、相対(接触)するCLのEUナンバリング164位のスレオニン(T)
・CH1のEUナンバリング147位のリジン(K)と、相対(接触)するCLのEUナンバリング138位のアスパラギン(N)
・CH1のEUナンバリング147位のリジン(K)と、相対(接触)するCLのEUナンバリング123位のグルタミン酸(E)
・CH1のEUナンバリング175位のグルタミン(Q)と、相対(接触)するCLのEUナンバリング160位のグルタミン(Q)
・CH1のEUナンバリング213位のリジン(K)と、相対(接触)するCLのEUナンバリング123位のグルタミン酸(E)
なお、これら部位のナンバリングについては、Kabatらの文献(Kabat EA et al. 1991. Sequence of Proteins of Immunological Interest. NIH)を参考にしている。
また、本発明におけるEUナンバリングとして記載された番号は、EU numbering(Sequences of proteins of immunological interest, NIH Publication No.91-3242)にしたがって記載した ものである。なお本発明において、「EUナンバリングX位のアミノ酸残基」、「EUナンバリングX位のアミノ酸」(Xは任意の数)は、「EUナンバリングX位に相当するアミノ酸残基」、「EUナンバリングX位に相当するアミノ酸」と読みかえることも可能である。
(a)CH1に含まれるアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCLに含まれるアミノ酸残基であってEUナンバリング180位のアミノ酸残基、
(b)CH1に含まれるアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCLに含まれるアミノ酸残基であってEUナンバリング131位のアミノ酸残基、
(c)CH1に含まれるアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCLに含まれるアミノ酸残基であってEUナンバリング164位のアミノ酸残基、
(d)CH1に含まれるアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCLに含まれるアミノ酸残基であってEUナンバリング138位のアミノ酸残基、
(e)CH1に含まれるアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCLに含まれるアミノ酸残基であってEUナンバリング123位のアミノ酸残基、
(f)CH1に含まれるアミノ酸残基であってEUナンバリング175位のアミノ酸残基、及びCLに含まれるアミノ酸残基であってEUナンバリング160位のアミノ酸残基。
(g)CH1に含まれるアミノ酸残基であってEUナンバリング213位のアミノ酸残基、及びCLに含まれるアミノ酸残基であってEUナンバリング123位のアミノ酸残基。
(X)グルタミン酸(E)、アスパラギン酸(D)、
(Y)リジン(K)、アルギニン(R)、ヒスチジン(H)。
(1)重鎖可変領域に含まれるアミノ酸残基であって、EUナンバリング39位に相当するアミノ酸残基、
(2)軽鎖可変領域に含まれるアミノ酸残基であって、EUナンバリング38位に相当するアミノ酸残基、
(3)重鎖可変領域に含まれるアミノ酸残基であって、EUナンバリング45位に相当するアミノ酸残基、
(4)軽鎖可変領域に含まれるアミノ酸残基であって、EUナンバリング44位に相当するアミノ酸残基。
(X)グルタミン酸(E)、アスパラギン酸(D)、
(Y)リジン(K)、アルギニン(R)、ヒスチジン(H)。
(1)グルタミン(Q)、
(2)グルタミン(Q)、
(3)ロイシン(L)、
(4)プロリン(P)
である。従って本発明の好ましい態様においては、これらアミノ酸残基が改変(例えば、電荷アミノ酸への置換)に供される。なお、上記(1)~(4)のアミノ酸残基の種類は、必ずしも上記のアミノ酸残基に限定されず、該アミノ酸に相当する他のアミノ酸であってもよい。例えば、軽鎖可変領域上のEUナンバリング38位に相当するアミノ酸として、ヒトの場合、例えば、ヒスチジン(H)であってもよい。当業者は、公知文献等(例えば、J. Mol. Recognit. (2003) 16, 113-120)を参照することにより、軽鎖の任意の位置について、その位置に相当するアミノ酸残基の種類を知ることが可能であり、適宜、当該アミノ酸残基について改変(例えば、電荷アミノ酸へ置換)することができる。
別の観点においては、本発明は、(1)抗原結合ドメイン(2)Fcγ受容体に対する結合活性が低下しているFc領域を含むドメイン、及び(3)CD3結合ドメイン、を含むポリペプチド会合体を有効成分として含有する医薬組成物を提供する。又、本発明は当該会合体を有効成分として含有する細胞傷害を誘導する治療剤(細胞傷害誘導治療剤)、細胞増殖抑制剤および抗癌剤に関する。本発明の医薬組成物は、癌治療剤あるいは癌予防剤として用いることもできる。本発明の細胞傷害誘導治療剤、細胞増殖抑制剤および抗癌剤は、癌を罹患している対象または再発する可能性がある対象に投与されることが好ましい。
(1)抗原結合ドメイン、
(2)Fcγ受容体に対する結合活性が低下しているFc領域を含むドメイン、及び
(3)CD3結合ドメイン、
を含むポリペプチド会合体を有効成分として含有する、細胞傷害誘導治療剤、細胞増殖抑制剤および抗癌剤は、当該ポリペプチド会合体を対象に投与する工程を含む癌を予防または治療する方法、または、細胞傷害誘導治療剤、細胞増殖抑制剤および抗癌剤の製造における当該ポリペプチド会合体の使用と表現することもできる。
また本発明は、本発明の方法に使用するための、本発明のポリペプチド会合体または本発明の製造方法により製造されたポリペプチド会合体に関する。
(1)概容
生体内に投与されたタンパク質の血中半減期を延ばす方法としては、目的タンパク質に抗体のFcドメインを付加し、FcRnを介したリサイクリング機能を利用する方法が良く知られている。しかしこの際、天然型のFcをBiTEに付加すると、一つの分子がBiTE部分の抗CD3 scFvを介してT細胞と結合すると同時に、Fc部分を介してNK細胞、マクロファージなどの細胞膜上のFcgR(Fcγ受容体)と結合することにより、癌抗原非依存的にこれらの細胞を架橋することによって活性化させ、各種サイトカインの誘導につながる可能性があるものと考えられた。そこで、BiTEにポリペプチドリンカーを介してFcγ受容体に対する結合活性が低下しているFc領域(サイレント型Fc)を連結させた分子、ERY2が作製され、この活性をBiTEと比較する検証が行われた。肝臓癌細胞において高発現していることが知られているGPIアンカータンパクであるGlypican 3(GPC3)に対する抗体のscFvとCD3 epsilonに対する抗体のscFvを短いペプチドリンカーで連結することによって、GPC3に対するBiTE(GPC3 BiTE)が作製された(図17A)。ついで、これにサイレント型Fcを連結したGPC3に対するERY2(GPC3 ERY2)が作製された(図17C)。また、比較として通常のIgG型抗GPC3抗体が作製された。この際、IgG型抗GPC3抗体は、ADCC活性がより増強することが知られている、糖鎖部分においてフコース含量を低減させた抗体、すなわち低フコース型抗体として調製された。
抗GPC3抗体の発現ベクターを鋳型として用いることによって、PCR法により増幅されたH鎖可変領域(anti-GPC3 VH)、L鎖可変領域(anti-GPC3 VL)をコードするcDNAがそれぞれ取得された。適切な配列を付加したプライマー及び当該cDNAを鋳型として用いたPCR法により、anti-GPC3 VHとanti-GPC3 VLとがGly・Gly・Gly・Gly・Ser(配列番号:7)を3回繰り返す配列からなるリンカーによって連結されたアミノ酸配列を有するanti-GPC3 scFvをコードするcDNA断片が作製された。
上記した方法と同様の適切な配列が付加されたプライマーを用いたPCR法、およびQuikChange Site-Directed Mutagenesis Kit(Stratagene社)を用いた方法等の当業者において公知の方法によって、GPC3 ERY2_Hk(配列番号:34、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、およびGPC3 ERY2_Hh(配列番号:35、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)をそれぞれコードするポリヌクレオチドが挿入された発現ベクターが作製された。
抗GPC3抗体(WO2006/006693においてヒト化GC33抗体として記載されている)の発現ベクターがエレクトロポレーション法によってGDPフコースノックアウトCHO細胞DXB11株(Cancer Sci. (2010) 101(10), 2227-33)に遺伝子導入された。限界希釈後に0.5 mg/mL Geneticine存在下で培養することにより薬剤耐性株が選択され、低フコース型抗GPC3抗体発現株が得られた。この細胞を培養して調製した培養上清より、Hitrap(R) ProteinA(Pharmacia社)を用いた通常のアフィニティー精製により抗体画分が調製された。次に当該抗体画分がSuperdex 20026/60(Pharmacia社)を用いたゲルろ過精製に供され、溶出液のモノマー画分を分取することによって低フコース型GPC3抗体が得られた。
(5-1)ヒト末梢血単核球(PBMC)溶液の調製
1,000単位/mLのヘパリン溶液(ノボ・ヘパリン注5千単位,ノボ・ノルディスク社)をあらかじめ100μL注入した注射器を用い、健常人ボランティア(成人)より末梢血50 mLが採取された。PBS(-)で2倍希釈した後に4等分された末梢血が、15 mLのFicoll-Paque PLUSをあらかじめ注入して遠心操作が行なわれたLeucosepリンパ球分離管(Cat. No. 227290、Greiner bio-one社)に加えられた。当該分離管の遠心分離(2,150 rpm、10分間、室温)の後、単核球画分層が分取された。10%FBSを含むDulbecco’s Modified Eagle’s Medium(SIGMA社、以下10%FBS/D-MEM)で1回単核球画分の細胞が洗浄された後、当該細胞は10%FBS/D-MEMを用いてその細胞密度が4×106 /mLに調製された。このように調製された細胞溶液がヒトPBMC溶液として以後の試験に用いられた。
細胞傷害活性はxCELLigenceリアルタイムセルアナライザー(ロシュ・ダイアグノスティックス社)を用いた細胞増殖抑制率で評価された。標的細胞にはSK-HEP-1細胞株にヒトGPC3を強制発現させて樹立したSK-pca13a細胞株が用いられた。SK-pca13aをディッシュから剥離し、1×104 cells/wellとなるようにE-Plate 96(ロシュ・ダイアグノスティックス社)プレートに100μL/wellで播き、xCELLigenceリアルタイムセルアナライザーを用いて生細胞の測定が開始された。翌日xCELLigenceリアルタイムセルアナライザーからプレートを取り出し、当該プレートに各濃度(0.004、0.04、0.4、4 nM)に調製した各抗体50μLが添加された。室温にて15分間反応させた後に(5-1)で調製されたヒトPBMC溶液50μL(2×105 cells/well)が加えられ、xCELLigenceリアルタイムセルアナライザーに当該プレートを再セットすることによって、生細胞の測定が開始された。反応は5%炭酸ガス、37℃条件下にて行われ、ヒトPBMC添加72時間後のCell Index値から、下式により細胞増殖抑制率(%)が求められた。なお計算に用いたCell Index値は、抗体添加直前のCell Index値が1となるようにノーマライズした後の数値が用いられた。
次に、癌抗原(GPC3)への結合ドメインを2価にすることにより、より癌細胞に対する結合活性を高めることで比活性を向上させることを試みた。GPC3 ERY2にさらにGPC3に対するscFvをもう一つ付加した形のGPC3 ERY5(図17D)、付加するGPC3に対する結合ドメインをscFvではなくFabの形にしたGPC3 ERY7(図17F)がそれぞれ作製された。また、GPC3 ERY5のCD3 epsilonに対するscFvを両腕に分離した形のGPC3 ERY6(図17E)も作製された。
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY5_Hh(配列番号:36、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY2_Hk
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY6_Hk(配列番号:37、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY6_Hh(配列番号:38、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY7_Hh(配列番号:39、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY7_L(配列番号:40、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY2_Hk
(1)GPC3 ERY8-2、GPC3 ERY9-1、GPC3 ERY10-1の作製
次に、BiTEの構造を持たずに目的の活性を持つ分子の創製が試みられた。癌抗原(GPC3)に対するIgGを基本骨格とし、これにCD3 epsilonに対するscFvを付加した形の分子が作製された。この際、基本骨格とするIgGのFcとしては、上述した場合と同様に、FcgR(Fcγ受容体)への結合性が減弱されたサイレント型Fcが用いられた。CD3 epsilonに対するscFvが抗GPC3抗体IgGのH鎖のN末に付加されたGPC3 ERY8-2(図17G)、H鎖のC末に付加されたGPC3 ERY10-1(図17I)、L鎖のC末に付加されたGPC3 ERY9-1(図17H)がそれぞれ作製された。
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY8-2_Hk(配列番号:41、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY8-2_Hh(配列番号:42、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY7_L
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY9-1_H(配列番号:43、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY9-1_L-His(配列番号:44、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY9-1_L-FLAG(配列番号:45、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY10-1_Hh(配列番号:46、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY8-2_Hk、GPC3 ERY7_L
(1)で記載されたin vitroのアッセイでGPC3 BiTEと同等以上の細胞傷害活性が認められたGPC3 ERY8-2、及び、GPC3 ERY10-1のin vivoの薬効の評価が行なわれた。GPC3を発現するヒト肺癌細胞株であるPC-10がヒトPBMCと混合されNOD scidマウスに移植された。当該マウスに対してGPC3 ERY8-2、あるいはGPC3 ERY10-1の投与による治療が行なわれた(pre-mixモデルと指称される)。
GPC3 ERY8-2、GPC3 ERY9-1、GPC3 ERY10-1等の一連の分子が、GPC3 BiTEに比較して顕著に長い血漿中半減期を有するか否かを検証するために、癌細胞が移植されていないNOD scidマウスに対して30μg/匹で投与された、GPC3 ERY9-1、GPC3 ERY10-1の血漿中濃度が経時的に測定された。
(4-1)FcgR結合型Fcを持つGPC3 ERY15-1の作製
GPC3 ERY8-2、GPC3 ERY9-1、GPC3 ERY10-1等の一連の分子が癌抗原非依存的なサイトカインの誘導を起こすか否かを検証するために、FcgR結合型Fcを持つGPC3 ERY15-1(図17J)を作製した。
GPC3 ERY15-1の癌抗原非依存的なサイトカイン誘導能が、GPC3 BiTE、GPC3 ERY9-1、GPC3 ERY10-1、及びcatumaxomabのそれと比較された。健常人ボランティアから採取した血液より上記の方法によりPBMCが調製された。ヒトPBMC溶液50 μL(2×105 細胞/ウェル)に、40 nMに調製された各抗体50μLが添加され、更に100μLの10%FBS/D-MEMが加えられた。反応液は5%炭酸ガス、37℃条件下にて培養された。72時間の培養の後に、培養上清が回収され、Human Th1/Th2/Th17 Kit(BD社)を用いたCytometric Beads Array(CBA)法にて培養上清中に分泌されたサイトカインが定量された。添付プロトコールに従った測定方法による試験はtriplicateにて行われた。
scFv構造と異なるCD3結合ドメインを持つ分子の検討が行われた。癌抗原(GPC3)に対するIgGの2本のH鎖のC末端にそれぞれCD3抗体のVH領域、VL領域を結合した分子型であるGPC3 ERY18(図17K)が作製された。この際、間のリンカー(Gly・Gly・Gly・Gly・Ser)の個数を1個~4個に変えた一連の分子(GPC3 ERY18 L1~L4)が作製された。また、適切な部位のアミノ酸をCysに置換してジスルフィド結合を導入することを可能とする分子(GPC3 ERY18 S1)も同時に作製された。
発現ベクター:GPC3 ERY18 L1_Hh(配列番号:49、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY18 L1_Hk(配列番号:50、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、及びGPC3 ERY7 L
発現ベクター:GPC3 ERY18 L2_Hh(配列番号:51、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY18 L2_Hk(配列番号:52、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、及びGPC3 ERY7 L
発現ベクター:GPC3 ERY18 L3_Hh(配列番号:53、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY18 L3_Hk(配列番号:54、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、及びGPC3 ERY7 L
発現ベクター:GPC3 ERY18 L4_Hh(配列番号:55、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY18 L4_Hk(配列番号:56、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、及びGPC3 ERY7 L
発現ベクター:GPC3 ERY18 S1_Hh(配列番号:57、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY18 S1_Hk(配列番号:58、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、及びGPC3 ERY7 L
次に、CD3結合ドメインがFab様構造である分子の検討が行われた。癌抗原(GPC3)に対するIgG抗体の2本のH鎖のC末端にそれぞれCD3抗体のVH領域とCH1領域、およびVL領域とCL領域が結合した分子型であるGPC3 ERY19-3が作製された(図17L)。すなわち、上記の方法と同様に適切な配列を付加したプライマーを用いたPCR法等の当業者にとって公知の方法により、GPC3 ERY19-3_Hh(配列番号:59、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、GPC3 ERY19-3_Hk(配列番号:60、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)をそれぞれコードするポリヌクレオチドが挿入された発現ベクターが作製された。
(1)概要
実施例3で調製されたGPC3 ERY10-1では、CH3ドメインの構造としてknobs-into-holeが用いられている。それぞれのH鎖のC末端にはHisタグとFLAGタグが付加されており、これらのタグを用いた2種類のアフィニティー精製を行うことにより、目的としている2種類のH鎖がヘテロ会合化したGPC3 ERY10-1分子が精製された。GPC3 ERY10-1分子を医薬品として製造する場合、GPC3 ERY10-1を発現する細胞の培養上精からプロテインAクロマトグラフィーを用いてFcドメインを有するポリペプチド会合体がまず精製される。さらに、HisタグアフィニティークロマトグラフィーとFLAGタグアフィニティークロマトグラフィーの2種類のクロマトグラムを用いた精製工程が必要となり、精製工程のコストが高くなるという課題がある。そこで本実施例では、HisタグとFLAGタグを用いることなく、プロテインAクロマトグラフィーのみを用いることによって目的とする2種類のH鎖がヘテロ会合化したGPC3 ERY10-1分子を精製することを可能とする分子改変の検討が行われた。
抗体H鎖可変領域として、GC33(2)H(抗ヒトGlypican-3抗体 H鎖可変領域、配列番号:61、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)をコードする遺伝子が当業者にとって公知の方法により作製された。同様に、抗体L鎖として、GC33-k0(抗ヒトGlypican-3抗体L鎖、配列番号:62、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)をコードする遺伝子が当業者にとって公知の方法により作製された。次に、抗体H鎖定常領域として、以下に示す遺伝子が当業者にとって公知の方法により作製された。
IgG1の配列中のEUナンバリング234番目および235番目のLeuがAlaに置換され、297番目のAsnがAlaに置換された変異が導入され、C末端のGly及びLysが除去されたLALA-G1d(配列番号:63、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)
LALA -G1d(配列番号:63、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)にCD3のscFv(抗ヒトCD3抗体H鎖可変領域及び抗ヒトCD3抗体L鎖可変領域がポリペプチドリンカーを介して結合されたもの)がC末端に結合されたLALA-G1d-CD3(配列番号:64、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)
LALA-G1d(配列番号:63、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)の配列中のEUナンバリング435番目のHisをArgに置換する変異及びEUナンバリング439番目のLysをGluに置換する変異が導入されたLALA-G3S3E-G1d(配列番号:65、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)
LALA-G1d-CD3(配列番号:64、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)の配列中のEUナンバリング356番目のAspをLysに置換する変異が導入されたLALA-S3K-G1d-CD3(配列番号:66、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)。
NTA1L/NTA1R/GC33-k0
NTA2L/NTA2R/GC33-k0
下記に示すポリペプチド会合体を含むFreeStyle293細胞の培養上清(以下CMと指称される)が試料として用いられた。
NTA1L/NTA1R/GC33-k0
NTA2L/NTA2R/GC33-k0
(1)GPC3 ERY 17-2及びGPC3 ERY 17-3の作製
次に、癌抗原(GPC3)に対するIgGを基本骨格とし、片方のFabをCD3 epsilonに対する結合ドメインに置き換えた形の分子が作製された。この際、基本骨格とするIgGのFcとしては、上述した場合と同様に、FcgR(Fcγ受容体)への結合性が減弱されたサイレント型Fcが用いられた。CD3 epsilonに対する結合ドメインとして、CD3 epsilonに対するFabのVHドメインとVLドメインを置き換えたGPC3 ERY17-2(図19A)と、CH1ドメインとCLドメインを置き換えたGPC3 ERY17-3(図19B)がそれぞれ作製された。
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY8-2_Hk、GPC3 ERY7_L、ERY17-2_Hh(配列番号:73、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、ERY17-2_L(配列番号:74、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:GPC3 ERY8-2_Hk、GPC3 ERY7_L、ERY17-3_Hh(配列番号:75、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、ERY17-3_L(配列番号:76、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)
得られた培養上清がAnti FLAG M2カラム(Sigma社)に添加され、当該カラムの洗浄の後、0.1 mg/mL FLAGペプチド(Sigma社)による溶出が実施された。目的分子を含む画分がHisTrap HPカラム(GE Healthcare社)に添加され、当該カラムの洗浄の後、イミダゾールの濃度勾配による溶出が実施された。目的分子を含む画分が限外ろ過によって濃縮された後、当該画分がSuperdex 200カラム(GE Healthcare社)に添加され、溶出液の単量体画分のみを回収することにより精製された各目的分子が得られた。
GPC3 ERY 17-2及びGPC3 ERY 17-3についてin vitroの細胞傷害活性が調べられた(図20)。その結果、いずれの分子も明らかにGPC3 BiTEを上回る細胞傷害活性を奏することが認められた。本発明において、癌抗原に対するIgGを基本骨格とし、片側のFabをCD3 epsilonに対する結合ドメインに置き換えた分子もBiTEと同等以上の細胞傷害活性を奏することが初めて明らかとなった。
in vitroのアッセイでGPC3 BiTEと同等以上の細胞傷害活性が認められたGPC3 ERY17-2のin vivoの薬効の評価がPC-10 T細胞移入モデルを用いて行なわれた。すなわち、GPC3 ERY17-2のPC-10 T細胞移入モデルによる薬効試験においては、下記のような試験が行われた。健常人ボランティアより採取した血液から分離されたPBMC及びT cell activation/ expansion kit/ human(MACS Miltenyi biotec社)を用いてT細胞の拡大培養が行なわれた。ヒト肺扁平上皮がん細胞株PC-10(免疫生物研究所) 1×107細胞と、マトリゲル基底膜マトリックス(BD社)が混和され、NOD scidマウス(日本クレア、雌、7W)のそけい部皮下に移植された。移植の日をday 0とした。マウスには移植前日、およびday 13、17、21、25に抗アシアロGM1抗体(和光純薬)が0.2 mg/匹で腹腔内に投与された。移植後13日目に腫瘍サイズと体重に応じて群分けが行なわれ、移植後14日目に前記拡大培養によって得られたT細胞が3×107細胞/匹で腹腔内に移植された。その2時間後から、GPC ERY17-2が30 μg/匹で静脈内投与された。GPC ERY17-2の投与はday 14、15、16、17、18に、計5回行われた。
(1)GPC3 ERY17-2-M20の作製
次に、CD3 epsilonに対する結合ドメインの配列が変わっても目的の活性を持つ分子の創製が試みられた。GPC3 ERY17-2のCD3 epsilonに対する結合ドメインの配列を変えたGPC3 ERY17-2-M20(図19A)が作製された。すなわち、CD3抗体(M20)の発現ベクターが鋳型として用いられ、上記した方法と同様の適切な配列を付加したプライマーを用いたPCR法等の当業者において公知の方法により、ERY17-2-M20_Hh(配列番号:77、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、ERY17-2-M20_L(配列番号:78、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)をそれぞれコードするポリヌクレオチドが挿入された一連の発現ベクターが作製された。
GPC3 ERY8-2_Hk、GPC3 ERY7_L、ERY17-2-M20_Hh(配列番号:77)、およびERY17-2-M20 _L(配列番号:78)の発現ベクターが共にFreeStyle293-F細胞に導入され、一過性にGPC3 ERY17-2-M20を発現させた。得られた培養上清が、φ0.22μmフィルターで濾過された後、平衡化したrProtein A Sepharose Fast Flowカラム(GE Healthcare)に負荷された。表3に示すバッファーにより洗浄1、2、および溶出1の各ステップが実施されることにより精製GPC3 ERY17-2-M20が得られた。
GPC3 ERY17-2-M20のin vitroの細胞傷害活性を調べたところ、GPC3 ERY17-2とほぼ同等の細胞傷害活性が認められた(図22)。このことからCD3 epsilonに対する結合ドメインの配列が変わった分子においても同等の細胞傷害活性を持つことが明らかとなった。
(1)EpCAM ERY17-2、EpCAM ERY17-3の作製
次に、標的とする癌抗原が変わっても目的の活性を持つ分子の創製が試みられた。GPC3 ERY17-2のGPC3に対するFabをEpCAMに対するFabに変えたEpCAM ERY17-2(図19A)と、GPC3 ERY17-3のGPC3に対するFabをEpCAMに対するFabに変えたEpCAM ERY17-3(図19B)がそれぞれ作製された。すなわち、EpCAM抗体の発現ベクターが鋳型として用いられ、上記した方法と同様の適切な配列を付加したプライマーを用いたPCR法等の当業者において公知の方法により、EpCAM ERY17_Hk(配列番号:79、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、EpCAM ERY17_L(配列番号:80、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)をそれぞれコードするポリヌクレオチドが挿入された一連の発現ベクターが作製された。
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:EpCAM ERY17_Hk(配列番号:79、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、EpCAM ERY17_L(配列番号:80、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、ERY17-2_Hh、ERY17-2_L
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:EpCAM ERY17_Hk、EpCAM ERY17_L、ERY17-3_Hh、ERY17-3_L
得られた培養上清がAnti FLAG M2カラム(Sigma社)に添加され、当該カラムの洗浄の後、0.1 mg/mL FLAGペプチド(Sigma社)による溶出が実施された。目的分子を含む画分がHisTrap HPカラム(GE Healthcare社)に添加され、当該カラムの洗浄の後、イミダゾールの濃度勾配による溶出が実施された。目的分子を含む画分が限外ろ過によって濃縮された後、当該画分がSuperdex 200カラム(GE Healthcare社)に添加され、溶出液の単量体画分のみを回収することにより精製された各目的分子が得られた。
EpCAM ERY17-2、EpCAM ERY17-3のin vitroの細胞傷害活性を調べたところ、いずれの分子においても強い細胞傷害活性が認められた(図23)。本発明において、癌抗原に対するIgGを基本骨格とし、片側のFabをCD3 epsilonに対する結合ドメインに置き換えた分子において、癌抗原の種類を変えても細胞傷害活性を持つことが明らかとなった。
(1)二重特異性抗体の設計
二重特異性抗体のそれぞれのCH1とCLドメインに変異を導入し、CH1/CLの界面の電荷的な反発を利用し、CH1/CLの界面会合を制御することによって、GPC3に対するH鎖とL鎖のみが会合し、CD3に対するH鎖とL鎖のみがそれぞれ特異的に会合させることが可能であると考えられた。電荷的な反発を利用してCH1/CL界面会合の制御を行うために、H鎖のCH1、またはL鎖のCL中のアミノ酸残基が、正電荷であるLys、または負電荷であるGluに置換された。
Anti-CD3抗体であるM12 (H鎖、配列番号:81およびL鎖、配列番号:82)と、Anti-GPC3抗体であるGC33(2)(H鎖、配列番号:83およびL鎖、配列番号:84)にCH1/CL界面会合制御が導入され、さらにH鎖同士の会合を避けるため、Knob into Hole(KiH)(WO1996/027011、Ridgway JB ら(Protein Engineering (1996) 9, 617-621)、Merchant AM ら (Nat. Biotechnol. (1998) 16, 677-681))の改変が導入された二重特異性抗体が作製された(図24A)。対照として、CH1/CL界面会合制御もKnob into Hole(KiH)改変も導入されていない二重特異性抗体も作製された(図24B)。具体的には、M12のH鎖(配列番号:81)のCH1の数アミノ酸がLysに置換されたM12_TH2h(配列番号:85)、L鎖(配列番号:82)のCLの数アミノ酸がGluに置換されたM12_TL17(配列番号:86)をそれぞれコードするポリヌクレオチドが挿入された発現ベクターが当業者にとって公知の方法により作製された。同様に、GC33(2)のH鎖(配列番号:83)のCH1の数アミノ酸がGluに置換されたGC33(2)_TH13k(配列番号:87)、GC33(2)_TH15k(配列番号:88)、L鎖(配列番号:84)のCLの数アミノ酸がLysに置換されたGC33(2)_TL16(配列番号:89)、GC33(2)_TL19(配列番号:90)をそれぞれコードするポリヌクレオチドが挿入された発現ベクターが当業者にとって公知の方法により作製された。
発現ベクター:M12_TH2h(配列番号:85)、M12_TL17(配列番号:86)、GC33(2)_TH13k(配列番号:87)、及びGC33(2)_TL16(配列番号:89)
発現ベクター:M12_TH2h(配列番号:85)、M12_TL17(配列番号:86)、GC33(2)_TH15k(配列番号:88)、及びGC33(2)_TL19(配列番号:90)
発現ベクター:M12のH鎖(配列番号:81)、M12のL鎖(配列番号:82)、GC33(2) のH鎖(配列番号:83)、及びGC33(2) のL鎖(配列番号:84)
GM1、GM2、GM0の各ポリペプチド会合体のin vitroの細胞傷害活性を調べたところ、GM1およびGM2は同等の細胞傷害活性を示すことが認められ、またその活性は明らかにGM0の活性を上回る細胞傷害活性であった(図25)。本発明において、CH1/CL界面制御の導入とKiHの改変を組み合わせることによって効率よく二重特異性抗体が作製されることが明らかとなった。
(1)EGFR ERY17-2の作製
さらに他の癌抗原を標的とした目的の活性を持つ分子の創製が試みられた。GPC3 ERY17-2のGPC3に対するFabをEGFRに対するFabに変えたEGFR ERY17-2(図19A)が作製された。すなわち、EGFR抗体の発現ベクターが鋳型として用いられ、上記した方法と同様の適切な配列を付加したプライマーを用いたPCR法等の当業者において公知の方法により、EGFR ERY17_Hk(配列番号:91、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、EGFR ERY17_L(配列番号:92、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)をそれぞれコードするポリヌクレオチドが挿入された一連の発現ベクターが作製された。
発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:EGFR ERY17_Hk(配列番号:91、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、EGFR ERY17_L(配列番号:92、シグナル配列であるアミノ末端19アミノ酸は成熟配列には含まれない)、ERY17-2_Hh、ERY17-2_L
得られた培養上清がAnti FLAG M2カラム(Sigma社)に添加され、当該カラムの洗浄の後、0.1 mg/mL FLAGペプチド(Sigma社)による溶出が実施された。目的分子を含む画分がHisTrap HPカラム(GE Healthcare社)に添加され、当該カラムの洗浄の後、イミダゾールの濃度勾配による溶出が実施された。目的分子を含む画分が限外ろ過によって濃縮された後、当該画分がSuperdex 200カラム(GE Healthcare社)に添加され、溶出液の単量体画分のみを回収することにより精製された各目的分子が得られた。
EGFR ERY17-2のin vitroの細胞傷害活性を調べたところ、強い細胞傷害活性が認められた(図26)。本発明において、癌抗原に対するIgGを基本骨格とし、片側のFabをCD3 epsilonに対する結合ドメインに置き換えた分子において、GPC3、EpCAMのみならず、癌抗原の種類をさらに変えても細胞傷害活性を持つことが明らかとなった。
Claims (68)
- 下記のドメイン;
(1)抗原結合ドメイン、
(2)Fcγ受容体に対する結合活性が低下しているFc領域を含むドメイン、及び、
(3)T細胞受容体複合体結合ドメイン、
を含むポリペプチド会合体。 - T細胞受容体複合体結合ドメインがT細胞受容体結合ドメインである、請求項1に記載のポリペプチド会合体。
- T細胞受容体複合体結合ドメインがCD3結合ドメインである、請求項1に記載のポリペプチド会合体。
- 抗原結合ドメインが二価の抗原結合ドメインである、請求項1から3のいずれかに記載のポリペプチド会合体。
- 二価の抗原結合ドメインがF(ab’)2の構造を有するドメインである、請求項4に記載のポリペプチド会合体。
- F(ab’)2の構造を有するドメインの重鎖定常領域を構成する二つのポリペプチドがFc領域を構成する二つのポリペプチドの各々に連結された、請求項5に記載のポリペプチド会合体。
- CD3結合ドメインがFc領域を構成する一つ又は二つのCH3に連結された、請求項6に記載のポリペプチド会合体。
- CD3結合ドメインを構成する重鎖Fv断片がFc領域を構成する一方のCH3に連結され、CD3結合ドメインを構成する軽鎖Fv断片がFc領域を構成するもう一方のCH3に連結された、請求項7に記載のポリペプチド会合体。
- CD3結合ドメインを構成する重鎖Fv断片に抗体のCH1ドメイン、及び、軽鎖Fv断片に抗体のCLドメインが連結された、請求項8に記載のポリペプチド会合体。
- CD3結合ドメインがF(ab’)2を構成する一つ又は二つのCLに連結された、請求項6に記載のポリペプチド会合体。
- CD3結合ドメインがF(ab’)2を構成する一つ又は二つのVHに連結された、請求項6に記載のポリペプチド会合体。
- CD3結合ドメインがF(ab’)2を構成する一つ又は二つのVLに連結された、請求項6に記載のポリペプチド会合体。
- CD3結合ドメインがFvである、請求項1から12のいずれかに記載のポリペプチド会合体。
- CD3結合ドメインがFabである、請求項1から7及び10から12のいずれかに記載のポリペプチド会合体。
- CD3結合ドメインがscFvである、請求項1から7及び10から12のいずれかに記載のポリペプチド会合体。
- CD3結合ドメインが一価である、請求項1から15のいずれかに記載のポリペプチド会合体。
- 抗原結合ドメインが一価のscFv及び一価のFabである、請求項1から3のいずれかに記載のポリペプチド会合体。
- 一価のscFvがCD3結合ドメインを構成するscFvを介してFc領域を構成する一つのポリペプチドに、一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一つのポリペプチドに各々連結され、当該Fabの軽鎖Fv断片がCL領域と連結された、請求項17に記載のポリペプチド会合体。
- 抗原結合ドメインが二価のscFvである、請求項1から3のいずれかに記載のポリペプチド会合体。
- 一価のscFvがCD3結合ドメインを構成する重鎖Fv断片を介してFc領域を構成する一つのポリペプチドに、他方の一価のscFvがCD3結合ドメインを構成する軽鎖Fv断片を介してFc領域を構成する他方の一つのポリペプチドに連結された、請求項19に記載のポリペプチド会合体。
- 一価のscFvがCD3結合ドメインを構成するscFvを介してFc領域を構成する一つのポリペプチドに、他方の一価のscFvがFc領域を構成する他方の一つのポリペプチドに連結された、請求項19に記載のポリペプチド会合体。
- 抗原結合ドメイン、及び、T細胞受容体複合体結合ドメインが各々一価のFabである、請求項1から3のいずれかに記載のポリペプチド会合体。
- 抗原結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、T細胞受容体結合ドメインを構成するFabの重鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結された、請求項22に記載のポリペプチド会合体。
- 抗原結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、T細胞受容体結合ドメインを構成するFabの軽鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの重鎖Fv断片がCL領域と連結された、請求項22に記載のポリペプチド会合体。
- 抗原結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、T細胞受容体結合ドメインを構成するFabの重鎖Fv断片がCL領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCH1領域と連結された、請求項22に記載のポリペプチド会合体。
- T細胞受容体結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、抗原結合ドメインを構成するFabの軽鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの重鎖Fv断片がCL領域と連結された、請求項22に記載のポリペプチド会合体。
- T細胞受容体結合ドメインを構成する一価のFabの重鎖Fv断片がCH1領域を介してFc領域を構成する一方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCL領域と連結され、抗原結合ドメインを構成するFabの重鎖Fv断片がCL領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fabの軽鎖Fv断片がCH1領域と連結された、請求項22に記載のポリペプチド会合体。
- (1)抗原に結合する一価のFab構造の重鎖Fv断片がCH1領域を介して前記Fc領域を構成する一方のポリペプチドに連結され、当該Fab構造の軽鎖Fv断片がCL領域と連結された抗原結合ドメイン、及び、
(2)T細胞受容体複合体に結合する一価のFab構造の重鎖Fv断片がCH1領域を介してFc領域を構成する他方のポリペプチドに連結され、当該Fab構造の軽鎖Fv断片がCL領域と連結されたT細胞受容体複合体結合ドメイン、
を含むポリペプチド会合体であって、抗原結合ドメイン中の重鎖Fv断片と抗原結合ドメイン中の軽鎖Fv断片またはT細胞受容体結合ドメイン中の重鎖Fv断片とT細胞受容体結合ドメイン中の軽鎖Fv断片が会合するようにCH1領域とCL領域の電荷が制御されている、請求項22に記載のポリペプチド会合体。 - T細胞受容体複合体結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基および抗原結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有する、請求項28に記載のポリペプチド会合体。
- 抗原結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基およびT細胞受容体複合体結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有する、請求項28に記載のポリペプチド会合体。
- T細胞受容体複合体結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基および抗原結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有し、抗原結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基およびT細胞受容体複合体結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに同種の電荷を有する、請求項28に記載のポリペプチド会合体。
- T細胞受容体複合体結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基およびT細胞受容体結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基が互いに異種の電荷を有する、請求項29又は31に記載のポリペプチド会合体。
- 抗原結合ドメイン中の重鎖Fv断片に連結されたCH1領域のアミノ酸残基および抗原結合ドメイン中の軽鎖Fv断片に連結されたCL領域のアミノ酸残基がともに異種の電荷を有する、請求項30または31に記載のポリペプチド会合体。
- T細胞受容体複合体結合ドメインがT細胞受容体結合ドメインである、請求項22から33のいずれかに記載のポリペプチド会合体。
- T細胞受容体結合ドメインがCD3結合ドメインである、請求項34に記載のポリペプチド会合体。
- CH1領域のアミノ酸残基およびCL領域のアミノ酸残基が、以下の(a)~(f)に示される1組又は2組以上のアミノ酸残基の組からなる群;
(a)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング180位のアミノ酸残基、
(b)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング131位のアミノ酸残基
(c)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング164位のアミノ酸残基
(d)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング138位のアミノ酸残基
(e)CH1領域のアミノ酸残基であってEUナンバリング147位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング123位のアミノ酸残基
(f)CH1領域のアミノ酸残基であってEUナンバリング175位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング160位のアミノ酸残基
より選択され、CH1領域のアミノ酸残基とCL領域のアミノ酸残基とが互いに異種の電荷を有するアミノ酸残基である、請求項32又は33に記載のポリペプチド会合体。 - さらに、以下の(g)に示されるアミノ酸残基の組を含む群より選択される、請求項36に記載のポリペプチド会合体。
(g)CH1領域のアミノ酸残基であってEUナンバリング213位のアミノ酸残基、及びCL領域のアミノ酸残基であってEUナンバリング123位のアミノ酸残基 - 前記異種の電荷を有するアミノ酸残基が、以下の(X)または(Y)のいずれかの群;
(X)グルタミン酸(E)、アスパラギン酸(D);
(Y)リジン(K)、アルギニン(R)、ヒスチジン(H);
に含まれるアミノ酸残基から選択される、請求項36又は37に記載のポリペプチド会合体。 - 前記異種の電荷を有するアミノ酸残基が、CH1領域のアミノ酸残基であってEUナンバリング175位のアミノ酸残基がLys、CL領域のアミノ酸残基であってEUナンバリング180位、131位及び160位のアミノ酸残基がいずれもGluである、請求項36から38のいずれかに記載のポリペプチド会合体。
- 前記異種の電荷を有するアミノ酸残基が、CH1領域のアミノ酸残基であってEUナンバリング147位及び175位のアミノ酸残基がGlu、CL領域のアミノ酸残基であってEUナンバリング180位、131位及び160位のアミノ酸残基がいずれもLysである、請求項36から38のいずれかに記載のポリペプチド会合体。
- さらに、CH1領域のアミノ酸残基であってEUナンバリング213位のアミノ酸残基がGluであり、CL領域のアミノ酸残基であってEUナンバリング123位のアミノ酸残基がLysである、請求項40に記載のポリペプチド会合体。
- Fc領域がFcγI、FcγIIA、FcγIIB、FcγIIIA及び/又はFcγIIIBのいずれかのFcγ受容体に対する結合活性が低下しているFc領域である請求項1から41のいずれかに記載のポリペプチド会合体。
- Fc領域が、配列番号:23に記載のFc領域、配列番号:24に記載のFc領域、配列番号:25に記載のFc領域、又は配列番号:26に記載のFc領域を構成するアミノ酸が変異しているFc領域であることを特徴とする、請求項1から42のいずれかに記載のポリペプチド会合体。
- Fc領域を構成するアミノ酸のうちEUナンバリングに従って特定される下記のいずれかのアミノ酸;
118位から260位のアミノ酸配列が配列番号:24に記載の配列、261位から447位のアミノ酸配列が配列番号:26に記載の配列であるFc領域である、請求項43に記載のポリペプチド会合体。 - Fc領域を構成するアミノ酸のうちEUナンバリングに従って特定される下記のいずれかのアミノ酸;
220位、226位、229位、231位、232位、233位、234位、235位、236位、237位、238位、239位、240位、264位、265位、266位、267位、269位、270位、295位、296位、297位、298位、299位、300位、325位、327位、328位、329位、330位、331位、332位、が変異しているFc領域である、請求項43に記載のポリペプチド会合体。 - Fc領域が配列番号:23に記載のFc領域を構成するアミノ酸が変異しているFc領域であることを特徴とする、請求項45に記載のポリペプチド会合体。
- Fc領域を構成するアミノ酸のうちEUナンバリングに従って特定される下記のいずれかのアミノ酸;
233位、234位、235位、236位、237位、327位、330位、331位、
が対応するIgG2またはIgG4においてそのEUナンバリングが対応するアミノ酸に置換されたFc領域である、請求項46に記載のポリペプチド会合体。 - Fc領域を構成するアミノ酸のうち、EUナンバリングに従って特定される下記のいずれかのアミノ酸;
234位、235位、297位、
が変異しているFc領域であることを特徴とする、請求項46に記載のポリペプチド会合体。 - 234位のアミノ酸がアラニン、235位のアミノ酸がアラニン、及び/又は、297位のアミノ酸がアラニンに変異していることを特徴とする、請求項48に記載のポリペプチド会合体。
- Fc領域を構成する二つのポリペプチドの配列が互いに異なる配列を有することを特徴とする、請求項43から49のいずれかに記載のポリペプチド会合体。
- Fc領域を構成する二つのポリペプチドの一方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される349位のアミノ酸がシステイン、366位のアミノ酸がトリプトファンに、他方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される356位のアミノ酸がシステイン、366位のアミノ酸がセリンに、368位のアミノ酸がアラニンに、407位のアミノ酸がバリンに変異していることを特徴とする、請求項1から50のいずれかに記載のポリペプチド会合体。
- Fc領域を構成する二つのポリペプチドの一方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される356位のアミノ酸がリジンに、他方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される439位のアミノ酸がグルタミン酸に変異し、いずれか一方のポリペプチドのアミノ酸残基のうちEUナンバリングに従って特定される435位のアミノ酸がアルギニンに変異していることを特徴とする、請求項1から50のいずれかに記載のポリペプチド会合体。
- Fc領域を構成する二つのポリペプチドのカルボキシ末端に存在する配列GKが欠失していることを特徴とする、請求項51又は52に記載のポリペプチド会合体。
- 抗原結合ドメインが同一のエピトープに結合する、請求項1から53のいずれかに記載のポリペプチド会合体。
- 同一のエピトープが配列番号:2に記載のアミノ酸配列からなるタンパク質中に存在する、請求項54に記載のポリペプチド会合体。
- 同一のエピトープが配列番号:4に記載のアミノ酸配列からなるタンパク質中に存在する、請求項54に記載のポリペプチド会合体。
- 抗原結合ドメインが互いに異なるエピトープに結合する、請求項1から53のいずれかに記載のポリペプチド会合体。
- 異なるエピトープが配列番号:2に記載のアミノ酸配列からなるタンパク質中に存在する、請求項57に記載のポリペプチド会合体。
- 異なるエピトープが配列番号:4に記載のアミノ酸配列からなるタンパク質中に存在する、請求項57に記載のポリペプチド会合体。
- 請求項1から59のいずれかに記載のポリペプチド会合体をコードするポリヌクレオチド。
- 請求項60に記載のポリヌクレオチドを含むベクター。
- 請求項61に記載のベクターを保持する細胞。
- 請求項62に記載の細胞を培養し培養上清からポリペプチド会合体を回収することを含むポリペプチド会合体の製造方法。
- 請求項1から59のいずれかに記載のポリペプチド会合体を有効成分として含む細胞傷害誘導治療剤。
- 細胞傷害誘導治療剤が癌治療剤である、請求項64に記載の治療剤。
- 癌が肝癌又は肺癌である、請求項65に記載の治療剤。
- 請求項1から59のいずれかに記載のポリペプチド会合体を治療が必要な患者に投与することを特徴とする、癌の治療又は予防方法。
- 癌が肝癌又は肺癌である、請求項67に記載の治療又は予防方法。
Priority Applications (30)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2819530A CA2819530C (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
EP23208888.0A EP4303237A3 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
EP18192844.1A EP3434767A1 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
PL11845786T PL2647707T3 (pl) | 2010-11-30 | 2011-11-30 | Środek terapeutyczny wywołujący cytotoksyczność |
CN201180068471.0A CN103429737B (zh) | 2010-11-30 | 2011-11-30 | 细胞毒诱导治疗剂 |
SG2013041587A SG190726A1 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
KR1020227018833A KR20220082104A (ko) | 2010-11-30 | 2011-11-30 | 세포상해 유도 치료제 |
US13/990,088 US11066483B2 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
EP11845786.0A EP2647707B1 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
JP2012546900A JP6278598B2 (ja) | 2010-11-30 | 2011-11-30 | 細胞傷害誘導治療剤 |
DK11845786.0T DK2647707T3 (en) | 2010-11-30 | 2011-11-30 | CYTOTOXICITY-INducing THERAPEUTIC AGENT |
KR1020217009395A KR102447595B1 (ko) | 2010-11-30 | 2011-11-30 | 세포상해 유도 치료제 |
RU2013129694A RU2663123C2 (ru) | 2010-11-30 | 2011-11-30 | Индуцирующий цитотоксичность терапевтический агент |
SI201131599T SI2647707T1 (sl) | 2010-11-30 | 2011-11-30 | Citotoksično-inducirajoče terapevtsko sredstvo |
KR1020137016886A KR101960109B1 (ko) | 2010-11-30 | 2011-11-30 | 세포상해 유도 치료제 |
KR1020247005207A KR20240025059A (ko) | 2010-11-30 | 2011-11-30 | 세포상해 유도 치료제 |
EP23200969.6A EP4279512A3 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
LTEP11845786.0T LT2647707T (lt) | 2010-11-30 | 2011-11-30 | Citotoksiškumą indukuojantis terapinis agentas |
ES11845786.0T ES2693232T3 (es) | 2010-11-30 | 2011-11-30 | Agente terapéutico que induce citotoxicidad |
EP23200956.3A EP4279511A3 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
AU2011337697A AU2011337697B2 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
KR1020197006956A KR102244173B1 (ko) | 2010-11-30 | 2011-11-30 | 세포상해 유도 치료제 |
EP23208881.5A EP4303236A3 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
EP23200977.9A EP4279513A3 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
MX2013006100A MX349057B (es) | 2010-11-30 | 2011-11-30 | Agente terapeutico que induce citotoxicidad. |
BR112013013311A BR112013013311A2 (pt) | 2010-11-30 | 2011-11-30 | agente terapêutico de indução de citotoxicidade |
AU2017200565A AU2017200565A1 (en) | 2010-11-30 | 2017-01-27 | Cytotoxicity-inducing therapeutic agent |
AU2017200999A AU2017200999B2 (en) | 2010-11-30 | 2017-02-14 | Cytotoxicity-inducing therapeutic agent |
HRP20181615TT HRP20181615T1 (hr) | 2010-11-30 | 2018-10-05 | Terapijsko sredstvo koje izaziva citotoksičnost |
US17/367,909 US20220041756A1 (en) | 2010-11-30 | 2021-07-06 | Cytotoxicity-inducing therapeutic agent |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010-266760 | 2010-11-30 | ||
JP2010266760 | 2010-11-30 | ||
JP2011121771 | 2011-05-31 | ||
JP2011-121771 | 2011-05-31 | ||
JP2011-238818 | 2011-10-31 | ||
JP2011238818 | 2011-10-31 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/990,088 A-371-Of-International US11066483B2 (en) | 2010-11-30 | 2011-11-30 | Cytotoxicity-inducing therapeutic agent |
US17/367,909 Division US20220041756A1 (en) | 2010-11-30 | 2021-07-06 | Cytotoxicity-inducing therapeutic agent |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012073985A1 true WO2012073985A1 (ja) | 2012-06-07 |
Family
ID=46171913
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2011/077603 WO2012073985A1 (ja) | 2010-11-30 | 2011-11-30 | 細胞傷害誘導治療剤 |
Country Status (23)
Country | Link |
---|---|
US (2) | US11066483B2 (ja) |
EP (7) | EP4303236A3 (ja) |
JP (6) | JP6278598B2 (ja) |
KR (5) | KR101960109B1 (ja) |
CN (2) | CN109160951A (ja) |
AR (1) | AR084053A1 (ja) |
AU (3) | AU2011337697B2 (ja) |
BR (1) | BR112013013311A2 (ja) |
CA (1) | CA2819530C (ja) |
DK (1) | DK2647707T3 (ja) |
ES (1) | ES2693232T3 (ja) |
HK (1) | HK1259128A1 (ja) |
HR (1) | HRP20181615T1 (ja) |
LT (1) | LT2647707T (ja) |
MX (1) | MX349057B (ja) |
PL (1) | PL2647707T3 (ja) |
PT (1) | PT2647707T (ja) |
RU (1) | RU2663123C2 (ja) |
SG (2) | SG190726A1 (ja) |
SI (1) | SI2647707T1 (ja) |
TR (1) | TR201815863T4 (ja) |
TW (6) | TWI736437B (ja) |
WO (1) | WO2012073985A1 (ja) |
Cited By (81)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014526895A (ja) * | 2011-08-23 | 2014-10-09 | ロシュ グリクアート アーゲー | 二重特異性抗原結合分子 |
CN104271602A (zh) * | 2012-11-21 | 2015-01-07 | 武汉友芝友生物制药有限公司 | 双特异性抗体 |
WO2015046467A1 (ja) | 2013-09-27 | 2015-04-02 | 中外製薬株式会社 | ポリペプチド異種多量体の製造方法 |
WO2015068847A1 (ja) | 2013-11-11 | 2015-05-14 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子 |
KR20150103008A (ko) * | 2012-11-28 | 2015-09-09 | 자임워크스 인코포레이티드 | 가공된 면역글로불린 중쇄-경쇄 쌍 및 이들의 용도 |
WO2015174439A1 (ja) * | 2014-05-13 | 2015-11-19 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子 |
WO2016047722A1 (ja) * | 2014-09-26 | 2016-03-31 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
WO2016076345A1 (ja) * | 2014-11-11 | 2016-05-19 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子のライブラリ |
US20160168247A1 (en) * | 2013-07-05 | 2016-06-16 | Genmab A/S | Humanized or chimeric cd3 antibodies |
WO2016182064A1 (ja) * | 2015-05-13 | 2016-11-17 | 中外製薬株式会社 | 多重抗原結合分子融合体、医薬組成物、線状エピトープの同定方法、および多重抗原結合分子融合体の製造方法 |
WO2016205200A1 (en) | 2015-06-16 | 2016-12-22 | Genentech, Inc. | Anti-cll-1 antibodies and methods of use |
WO2016131769A3 (en) * | 2015-02-16 | 2016-12-22 | Lonza Ltd | Cl and/or ch1 mutated antibodies for drug conjugation |
KR20170044613A (ko) * | 2014-05-28 | 2017-04-25 | 자임워크스 인코포레이티드 | 변형된 항원 결합 폴리펩티드 작제물 및 이의 용도 |
WO2017086419A1 (ja) * | 2015-11-18 | 2017-05-26 | 中外製薬株式会社 | 液性免疫応答の増強方法 |
WO2017086367A1 (ja) | 2015-11-18 | 2017-05-26 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子を用いた併用療法 |
US9670269B2 (en) | 2006-03-31 | 2017-06-06 | Chugai Seiyaku Kabushiki Kaisha | Methods of modifying antibodies for purification of bispecific antibodies |
US9676845B2 (en) | 2009-06-16 | 2017-06-13 | Hoffmann-La Roche, Inc. | Bispecific antigen binding proteins |
JP2017140051A (ja) * | 2012-10-04 | 2017-08-17 | リサーチ ディベロップメント ファウンデーション | セリンプロテアーゼ分子および療法 |
JP2017522888A (ja) * | 2014-07-29 | 2017-08-17 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | 多重特異性抗体 |
JP2017524740A (ja) * | 2014-07-26 | 2017-08-31 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | 二重特異性抗体のための精製プラットフォーム |
JP2017525690A (ja) * | 2014-08-04 | 2017-09-07 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | 二重特異性t細胞活性化抗原結合分子 |
WO2017159287A1 (ja) | 2016-03-14 | 2017-09-21 | 中外製薬株式会社 | 癌の治療に用いるための細胞傷害誘導治療剤 |
US9771573B2 (en) | 2012-10-03 | 2017-09-26 | Zymeworks Inc. | Methods of quantitating heavy and light chain polypeptide pairs |
JP2017532290A (ja) * | 2014-08-04 | 2017-11-02 | エンクマフ アーゲー | Cd3イプシロンおよびbcmaに対する二特異性抗体 |
US9828429B2 (en) | 2007-09-26 | 2017-11-28 | Chugai Seiyaku Kabushiki Kaisha | Method of modifying isoelectric point of antibody via amino acid substitution in CDR |
JPWO2016159213A1 (ja) * | 2015-04-01 | 2018-02-15 | 中外製薬株式会社 | ポリペプチド異種多量体の製造方法 |
US9914785B2 (en) | 2012-11-28 | 2018-03-13 | Zymeworks Inc. | Engineered immunoglobulin heavy chain-light chain pairs and uses thereof |
JP2018076304A (ja) * | 2012-06-14 | 2018-05-17 | 中外製薬株式会社 | 改変されたFc領域を含む抗原結合分子 |
US9982036B2 (en) | 2011-02-28 | 2018-05-29 | Hoffmann-La Roche Inc. | Dual FC antigen binding proteins |
JP2018093879A (ja) * | 2010-11-30 | 2018-06-21 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
US10011858B2 (en) | 2005-03-31 | 2018-07-03 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
US10087250B2 (en) | 2012-10-08 | 2018-10-02 | Roche Glycart Ag | Fc-free antibodies comprising two fab-fragments and methods of use |
US10106612B2 (en) | 2012-06-27 | 2018-10-23 | Hoffmann-La Roche Inc. | Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof |
US10138293B2 (en) | 2007-12-21 | 2018-11-27 | Hoffmann-La Roche, Inc. | Bivalent, bispecific antibodies |
US10155815B2 (en) | 2013-02-26 | 2018-12-18 | Roche Glycart Ag | Bispecific T cell activating antigen binding molecules |
US10174124B2 (en) | 2013-12-17 | 2019-01-08 | Genentech, Inc. | Anti-CD3 antibodies and methods of use |
EP2961773B1 (en) * | 2013-02-26 | 2019-03-20 | Roche Glycart AG | Bispecific t cell activating antigen binding molecules |
US10323094B2 (en) | 2015-06-16 | 2019-06-18 | Genentech, Inc. | Humanized and affinity matured antibodies to FcRH5 and methods of use |
US10323099B2 (en) | 2013-10-11 | 2019-06-18 | Hoffmann-La Roche Inc. | Multispecific domain exchanged common variable light chain antibodies |
WO2019160007A1 (ja) | 2018-02-14 | 2019-08-22 | 中外製薬株式会社 | 抗原結合分子および組合せ |
JP2019529499A (ja) * | 2016-09-30 | 2019-10-17 | ベイラー カレッジ オブ メディスンBaylor College Of Medicine | 組織指向性発現を用いた抗体ベースの遺伝子治療 |
WO2019244973A1 (ja) | 2018-06-20 | 2019-12-26 | 中外製薬株式会社 | 標的細胞に対する免疫反応を活性化する方法およびその組成物 |
US10550193B2 (en) * | 2014-03-19 | 2020-02-04 | Regeneron Pharmaceuticals, Inc. | Methods and antibody compositions for tumor treatment |
US10556952B2 (en) | 2015-03-30 | 2020-02-11 | Regeneron Pharmaceuticals, Inc. | Heavy chain constant regions with reduced binding to Fc gamma receptors |
US10596257B2 (en) | 2016-01-08 | 2020-03-24 | Hoffmann-La Roche Inc. | Methods of treating CEA-positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD3 bispecific antibodies |
US10611825B2 (en) | 2011-02-28 | 2020-04-07 | Hoffmann La-Roche Inc. | Monovalent antigen binding proteins |
US10662244B2 (en) | 2014-11-17 | 2020-05-26 | Regeneron Pharmaceuticals, Inc. | Methods for tumor treatment using CD3XCD20 bispecific antibody |
US20200199231A1 (en) * | 2015-01-08 | 2020-06-25 | Genmab A/S | Bispecific antibodies against cd3 and cd20 |
US10766967B2 (en) | 2015-10-02 | 2020-09-08 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
US10766960B2 (en) | 2012-12-27 | 2020-09-08 | Chugai Seiyaku Kabushiki Kaisha | Heterodimerized polypeptide |
US10781262B2 (en) | 2014-11-20 | 2020-09-22 | Hoffmann-La Roche Inc. | Combination therapy of T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists |
WO2020213724A1 (ja) | 2019-04-19 | 2020-10-22 | 中外製薬株式会社 | 抗体改変部位認識キメラ受容体 |
US10882918B2 (en) | 2016-09-30 | 2021-01-05 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
US10988537B2 (en) | 2013-02-01 | 2021-04-27 | Regeneren Pharmaceuticals, Inc. | Antibodies comprising chimeric constant domains |
US11013801B2 (en) | 2015-12-09 | 2021-05-25 | Hoffmann-La Roche Inc. | Treatment method |
US11046784B2 (en) | 2006-03-31 | 2021-06-29 | Chugai Seiyaku Kabushiki Kaisha | Methods for controlling blood pharmacokinetics of antibodies |
US11072656B2 (en) | 2012-09-21 | 2021-07-27 | Regeneran Pharmaceuticals, Inc. | Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof |
US11084877B2 (en) | 2014-09-12 | 2021-08-10 | Genentech, Inc. | Anti-CLL-1 antibodies and immunoconjugates |
JP2021159081A (ja) * | 2020-03-31 | 2021-10-11 | 中外製薬株式会社 | Dll3標的多重特異性抗原結合分子およびその使用 |
EP3130606B1 (en) | 2014-04-07 | 2021-10-13 | Chugai Seiyaku Kabushiki Kaisha | Immunoactivating bispecific antibodies |
US11161915B2 (en) | 2015-10-08 | 2021-11-02 | Zymeworks Inc. | Antigen-binding polypeptide constructs comprising kappa and lambda light chains and uses thereof |
US11242390B2 (en) | 2016-03-22 | 2022-02-08 | Hoffmann-La Roche Inc. | Protease-activated T cell bispecific molecules |
KR20220019706A (ko) | 2019-06-10 | 2022-02-17 | 추가이 세이야쿠 가부시키가이샤 | 사이토카인 저해제와 조합하여 사용하기 위한 항t세포 항원 결합 분자 |
US11332533B2 (en) | 2007-09-26 | 2022-05-17 | Chugai Seiyaku Kabushiki Kaisha | Modified antibody constant region |
US11421022B2 (en) | 2012-06-27 | 2022-08-23 | Hoffmann-La Roche Inc. | Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof |
US11466094B2 (en) | 2016-11-15 | 2022-10-11 | Genentech, Inc. | Dosing for treatment with anti-CD20/anti-CD3 bispecific antibodies |
KR20220161375A (ko) | 2020-03-31 | 2022-12-06 | 추가이 세이야쿠 가부시키가이샤 | 다중 특이성 항원 결합 분자를 제조하기 위한 방법 |
KR20220161156A (ko) | 2020-03-31 | 2022-12-06 | 추가이 세이야쿠 가부시키가이샤 | 면역 활성화 다중 특이성 항원 결합 분자 및 그의 사용 |
US11590223B2 (en) | 2018-08-31 | 2023-02-28 | Regeneron Pharmaceuticals, Inc. | Dosing strategy that mitigates cytokine release syndrome for therapeutic antibodies |
KR20230028225A (ko) | 2020-06-19 | 2023-02-28 | 추가이 세이야쿠 가부시키가이샤 | 혈관신생 저해제와 조합하여 사용하기 위한 항t세포 항원 결합 분자 |
US11618790B2 (en) | 2010-12-23 | 2023-04-04 | Hoffmann-La Roche Inc. | Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery |
US11639397B2 (en) | 2011-08-23 | 2023-05-02 | Roche Glycart Ag | Bispecific antibodies specific for T-cell activating antigens and a tumor antigen and methods of use |
US11649262B2 (en) | 2015-12-28 | 2023-05-16 | Chugai Seiyaku Kabushiki Kaisha | Method for promoting efficiency of purification of Fc region-containing polypeptide |
US11667713B2 (en) | 2017-12-28 | 2023-06-06 | Chugai Seiyaku Kabushiki Kaisha | Cytotoxicity-inducing therapeutic agent |
US11780920B2 (en) | 2020-06-19 | 2023-10-10 | Hoffmann-La Roche Inc. | Antibodies binding to CD3 and CD19 |
US11845805B2 (en) | 2020-09-10 | 2023-12-19 | Genmab A/S | Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma |
US11845799B2 (en) | 2019-12-13 | 2023-12-19 | Genentech, Inc. | Anti-Ly6G6D antibodies and methods of use |
US11851476B2 (en) | 2011-10-31 | 2023-12-26 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule having regulated conjugation between heavy-chain and light-chain |
US11858995B2 (en) | 2020-09-10 | 2024-01-02 | Genmab A/S | Bispecific antibodies against CD3 and CD20 for treating chronic lymphocytic leukemia |
US11866498B2 (en) | 2018-02-08 | 2024-01-09 | Genentech, Inc. | Bispecific antigen-binding molecules and methods of use |
US11952422B2 (en) | 2017-12-05 | 2024-04-09 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule comprising altered antibody variable region binding CD3 and CD137 |
Families Citing this family (47)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10053513B2 (en) * | 2009-11-30 | 2018-08-21 | Janssen Biotech, Inc. | Antibody Fc mutants with ablated effector functions |
RU2632647C2 (ru) | 2011-04-22 | 2017-10-06 | Аптево Рисерч Энд Девелопмент Ллс | Белки, связывающие специфический мембранный антиген простаты, и соответствующие композиции и способы |
EA201892339A3 (ru) | 2011-12-19 | 2019-06-28 | Дзе Рокфеллер Юниверсити | Несиалированный выделенный полипептид, способ получения указанного полипептида и фармацевтическая композиция для лечения воспалительного заболевания |
US11053316B2 (en) | 2013-01-14 | 2021-07-06 | Xencor, Inc. | Optimized antibody variable regions |
AU2014205086B2 (en) | 2013-01-14 | 2019-04-18 | Xencor, Inc. | Novel heterodimeric proteins |
US10858417B2 (en) | 2013-03-15 | 2020-12-08 | Xencor, Inc. | Heterodimeric proteins |
SG11201608054YA (en) | 2014-04-02 | 2016-10-28 | Hoffmann La Roche | Method for detecting multispecific antibody light chain mispairing |
UA117289C2 (uk) | 2014-04-02 | 2018-07-10 | Ф. Хоффманн-Ля Рош Аг | Мультиспецифічне антитіло |
EP3164417A1 (en) | 2014-07-01 | 2017-05-10 | Pfizer Inc. | Bispecific heterodimeric diabodies and uses thereof |
US9777073B2 (en) * | 2014-07-21 | 2017-10-03 | Wuhan Yzy Biopharma Co., Ltd. | Construction and application of bispecific antibody EpCAM×CD3 |
WO2016019969A1 (en) | 2014-08-08 | 2016-02-11 | Ludwig-Maximilians-Universität München | Subcutaneously administered bispecific antibodies for use in the treatment of cancer |
JP6708635B2 (ja) | 2014-10-09 | 2020-06-10 | エンクマフ エスアーエールエル | CD3εおよびROR1に対する二特異性抗体 |
EP3023437A1 (en) | 2014-11-20 | 2016-05-25 | EngMab AG | Bispecific antibodies against CD3epsilon and BCMA |
US10259887B2 (en) | 2014-11-26 | 2019-04-16 | Xencor, Inc. | Heterodimeric antibodies that bind CD3 and tumor antigens |
CN116333153A (zh) | 2014-11-26 | 2023-06-27 | 森科股份有限公司 | 结合cd3和肿瘤抗原的异二聚体抗体 |
PL3227332T3 (pl) | 2014-12-03 | 2020-06-15 | F. Hoffmann-La Roche Ag | Wielospecyficzne przeciwciała |
CA2978580A1 (en) | 2015-03-04 | 2016-09-09 | The Rockefeller University | Anti-inflammatory polypeptides |
TWI829617B (zh) | 2015-07-31 | 2024-01-21 | 德商安美基研究(慕尼黑)公司 | Flt3及cd3抗體構築體 |
TWI744242B (zh) | 2015-07-31 | 2021-11-01 | 德商安美基研究(慕尼黑)公司 | Egfrviii及cd3抗體構築體 |
TWI796283B (zh) | 2015-07-31 | 2023-03-21 | 德商安美基研究(慕尼黑)公司 | Msln及cd3抗體構築體 |
CN107969127B (zh) * | 2015-09-08 | 2022-09-06 | 赛瑞品股份有限公司 | Apoa-1融合多肽及相关组合物和方法 |
EA201890613A1 (ru) | 2015-09-21 | 2018-10-31 | Аптево Рисёрч Энд Девелопмент Ллс | Полипептиды, связывающие cd3 |
AR106188A1 (es) | 2015-10-01 | 2017-12-20 | Hoffmann La Roche | Anticuerpos anti-cd19 humano humanizados y métodos de utilización |
EP3150636A1 (en) | 2015-10-02 | 2017-04-05 | F. Hoffmann-La Roche AG | Tetravalent multispecific antibodies |
EP3150637A1 (en) | 2015-10-02 | 2017-04-05 | F. Hoffmann-La Roche AG | Multispecific antibodies |
US20170096485A1 (en) * | 2015-10-02 | 2017-04-06 | Hoffmann-La Roche Inc. | Bispecific t cell activating antigen binding molecules |
US11623957B2 (en) | 2015-12-07 | 2023-04-11 | Xencor, Inc. | Heterodimeric antibodies that bind CD3 and PSMA |
MX2018008934A (es) | 2016-01-22 | 2019-03-28 | Janssen Biotech Inc | Anticuerpos anti-ror1, anticuerpos biespecíficos ror1 x cd3 y métodos para su uso. |
KR20180103084A (ko) | 2016-02-03 | 2018-09-18 | 암젠 리서치 (뮌헨) 게엠베하 | Bcma 및 cd3 이중특이성 t 세포 맞물림 항체 작제물 |
EA039859B1 (ru) | 2016-02-03 | 2022-03-21 | Эмджен Рисерч (Мюник) Гмбх | Биспецифические конструкты антител, связывающие egfrviii и cd3 |
RU2767357C2 (ru) | 2016-06-14 | 2022-03-17 | Ксенкор, Инк. | Биспецифические антитела-ингибиторы контрольных точек |
DK3428194T3 (da) | 2017-07-14 | 2021-11-15 | Immatics Biotechnologies Gmbh | Forbedret polypeptidmolekyle med dobbelt specificitet |
CN112423785A (zh) | 2018-07-19 | 2021-02-26 | 瑞泽恩制药公司 | 双特异性抗BCMAx抗CD3抗体及其用途 |
WO2020086758A1 (en) * | 2018-10-23 | 2020-04-30 | Dragonfly Therapeutics, Inc. | Heterodimeric fc-fused proteins |
JP2022512798A (ja) * | 2018-10-25 | 2022-02-07 | エフ.ホフマン-ラ ロシュ アーゲー | 抗体FcRn結合の改変 |
JP7053441B2 (ja) | 2018-12-05 | 2022-04-12 | 三菱造船株式会社 | 船舶 |
CN112969476A (zh) * | 2018-12-07 | 2021-06-15 | 江苏恒瑞医药股份有限公司 | 多特异性蛋白分子 |
MX2021012872A (es) * | 2019-04-25 | 2021-11-17 | Hoffmann La Roche | Polipeptidos multiespecificos terapeuticos activados mediante intercambio de cadenas polipeptidicas. |
KR102560876B1 (ko) | 2019-06-11 | 2023-07-28 | 바이오아트라, 인코퍼레이티드 | 조건부 활성 항-epcam 항체, 항체 단편, 이들의 면역접합체 및 이의 용도 |
CN112111012B (zh) * | 2019-06-20 | 2023-07-04 | 成都恩沐生物科技有限公司 | 共价多特异性抗体 |
MX2022007404A (es) * | 2019-12-18 | 2022-09-19 | Janssen Biotech Inc | Materiales y metodos para direccionamiento biologico in vivo. |
CN111333720B (zh) * | 2020-03-16 | 2021-11-02 | 重庆理工大学 | 抗hpv16e7蛋白单抗79a11、杂交瘤细胞株及其制备方法和应用 |
WO2021231976A1 (en) | 2020-05-14 | 2021-11-18 | Xencor, Inc. | Heterodimeric antibodies that bind prostate specific membrane antigen (psma) and cd3 |
CA3192204A1 (en) | 2020-08-19 | 2022-02-24 | Xencor, Inc. | Anti-cd28 and/or anti-b7h3 compositions |
EP4228693A1 (en) | 2020-10-13 | 2023-08-23 | Janssen Biotech, Inc. | Bioengineered t cell mediated immunity, materials and other methods for modulating cluster of differentiation iv &/or viii |
IL305736A (en) | 2021-03-09 | 2023-11-01 | Xencor Inc | Heterodimeric antibodies that bind CD3 and CLDN6 |
EP4305065A1 (en) | 2021-03-10 | 2024-01-17 | Xencor, Inc. | Heterodimeric antibodies that bind cd3 and gpc3 |
Citations (35)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3773719A (en) | 1966-12-06 | 1973-11-20 | Hoffmann La Roche | 2-aminoxy-2'-acyl-acetanilide |
EP0058481A1 (en) | 1981-02-16 | 1982-08-25 | Zeneca Limited | Continuous release pharmaceutical compositions |
EP0133988A2 (de) | 1983-08-02 | 1985-03-13 | Hoechst Aktiengesellschaft | Regulatorische Peptide enthaltende pharmazeutische Präparate mit protrahierter Freisetzung und Verfahren zu deren Herstellung |
EP0239400A2 (en) | 1986-03-27 | 1987-09-30 | Medical Research Council | Recombinant antibodies and methods for their production |
WO1988001649A1 (en) | 1986-09-02 | 1988-03-10 | Genex Corporation | Single polypeptide chain binding molecules |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
WO1992001047A1 (en) | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1992003918A1 (en) | 1990-08-29 | 1992-03-19 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO1992020791A1 (en) | 1990-07-10 | 1992-11-26 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1993006213A1 (en) | 1991-09-23 | 1993-04-01 | Medical Research Council | Production of chimeric antibodies - a combinatorial approach |
WO1993011236A1 (en) | 1991-12-02 | 1993-06-10 | Medical Research Council | Production of anti-self antibodies from antibody segment repertoires and displayed on phage |
WO1993012227A1 (en) | 1991-12-17 | 1993-06-24 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO1993019172A1 (en) | 1992-03-24 | 1993-09-30 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US5260203A (en) | 1986-09-02 | 1993-11-09 | Enzon, Inc. | Single polypeptide chain binding molecules |
WO1994002602A1 (en) | 1992-07-24 | 1994-02-03 | Cell Genesys, Inc. | Generation of xenogeneic antibodies |
WO1994011523A2 (en) | 1992-11-13 | 1994-05-26 | Idec Pharmaceuticals Corporation | Fully impaired consensus kozac sequences for mammalian expression |
WO1994025585A1 (en) | 1993-04-26 | 1994-11-10 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO1995001438A1 (en) | 1993-06-30 | 1995-01-12 | Medical Research Council | Sbp members with a chemical moiety covalently bound within the binding site; production and selection thereof |
WO1995015388A1 (en) | 1993-12-03 | 1995-06-08 | Medical Research Council | Recombinant binding proteins and peptides |
WO1996002576A1 (fr) | 1994-07-13 | 1996-02-01 | Chugai Seiyaku Kabushiki Kaisha | Anticorps humain reconstitue contre l'interleukine-8 humaine |
WO1996027011A1 (en) | 1995-03-01 | 1996-09-06 | Genentech, Inc. | A method for making heteromultimeric polypeptides |
WO1996034096A1 (en) | 1995-04-28 | 1996-10-31 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
WO1996033735A1 (en) | 1995-04-27 | 1996-10-31 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
WO2003000883A1 (en) | 2001-06-22 | 2003-01-03 | Chugai Seiyaku Kabushiki Kaisha | Cell proliferation inhibitors containing anti-glypican 3 antibody |
WO2003104453A1 (ja) | 2002-06-05 | 2003-12-18 | 中外製薬株式会社 | 抗体作製方法 |
WO2004022754A1 (ja) | 2002-09-04 | 2004-03-18 | Chugai Seiyaku Kabushiki Kaisha | MRL/lprマウスを用いた抗体の作製 |
WO2004065611A1 (ja) | 2003-01-21 | 2004-08-05 | Chugai Seiyaku Kabushiki Kaisha | 抗体の軽鎖スクリーニング方法 |
WO2006006693A1 (ja) | 2004-07-09 | 2006-01-19 | Chugai Seiyaku Kabushiki Kaisha | 抗グリピカン3抗体 |
WO2006106905A1 (ja) | 2005-03-31 | 2006-10-12 | Chugai Seiyaku Kabushiki Kaisha | 会合制御によるポリペプチド製造方法 |
WO2006132352A1 (ja) | 2005-06-10 | 2006-12-14 | Chugai Seiyaku Kabushiki Kaisha | sc(Fv)2を含有する医薬組成物 |
WO2008090960A1 (ja) * | 2007-01-24 | 2008-07-31 | Kyowa Hakko Kirin Co., Ltd. | ガングリオシドgm2に特異的に結合する遺伝子組換え抗体組成物 |
WO2009011941A2 (en) | 2007-04-04 | 2009-01-22 | The Government Of U.S.A., As Represented By The Secretary, Departmetnt Of Health & Human Services | Monoclonal antibodies against dengue and other viruses with deletion in fc region |
WO2009041613A1 (ja) * | 2007-09-26 | 2009-04-02 | Chugai Seiyaku Kabushiki Kaisha | 抗体定常領域改変体 |
WO2009120922A2 (en) * | 2008-03-27 | 2009-10-01 | Zymogenetics, Inc. | Compositions and methods for inhibiting pdgfrbeta and vegf-a |
WO2010034441A1 (en) * | 2008-09-26 | 2010-04-01 | F. Hoffmann-La Roche Ag | Bispecific anti-egfr/anti-igf-1r antibodies |
Family Cites Families (163)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5912436B2 (ja) | 1980-08-05 | 1984-03-23 | ファナック株式会社 | 工業用ロボットの安全機構 |
US6121424A (en) | 1991-11-25 | 2000-09-19 | Enzon, Inc. | Multivalent antigen-binding proteins |
EP0307434B2 (en) | 1987-03-18 | 1998-07-29 | Scotgen Biopharmaceuticals, Inc. | Altered antibodies |
US5601819A (en) | 1988-08-11 | 1997-02-11 | The General Hospital Corporation | Bispecific antibodies for selective immune regulation and for selective immune cell binding |
DE69032484T4 (de) | 1989-10-27 | 1999-09-16 | Arch Dev Corp | Zusammensetzungen und deren verwendung zur förderung der immunopotentiation |
US5859205A (en) | 1989-12-21 | 1999-01-12 | Celltech Limited | Humanised antibodies |
GB9206422D0 (en) | 1992-03-24 | 1992-05-06 | Bolt Sarah L | Antibody preparation |
US6129914A (en) | 1992-03-27 | 2000-10-10 | Protein Design Labs, Inc. | Bispecific antibody effective to treat B-cell lymphoma and cell line |
UA40577C2 (uk) | 1993-08-02 | 2001-08-15 | Мерк Патент Гмбх | Біспецифічна молекула, що використовується для лізису пухлинних клітин, спосіб її одержання, моноклональне антитіло (варіанти), фармацевтичний препарат, фармацевтичний набір (варіанти), спосіб видалення пухлинних клітин |
US5595756A (en) | 1993-12-22 | 1997-01-21 | Inex Pharmaceuticals Corporation | Liposomal compositions for enhanced retention of bioactive agents |
DE4419399C1 (de) | 1994-06-03 | 1995-03-09 | Gsf Forschungszentrum Umwelt | Verfahren zur Herstellung von heterologen bispezifischen Antikörpern |
US5945311A (en) | 1994-06-03 | 1999-08-31 | GSF--Forschungszentrumfur Umweltund Gesundheit | Method for producing heterologous bi-specific antibodies |
US6805869B2 (en) | 1996-06-12 | 2004-10-19 | Shanghai Cp Guojian Pharmaceutical Co., Ltd. | Cellular vaccines and immunotherapeutics and methods for their preparation |
ATE210682T1 (de) * | 1996-09-03 | 2001-12-15 | Gsf Forschungszentrum Umwelt | Zerstörung von kontaminierenden tumorzellen in stammzelltransplantaten mit bispezifischen antikörpern |
IL132560A0 (en) | 1997-05-02 | 2001-03-19 | Genentech Inc | A method for making multispecific antibodies having heteromultimeric and common components |
US20030207346A1 (en) | 1997-05-02 | 2003-11-06 | William R. Arathoon | Method for making multispecific antibodies having heteromultimeric and common components |
US20020062010A1 (en) | 1997-05-02 | 2002-05-23 | Genentech, Inc. | Method for making multispecific antibodies having heteromultimeric and common components |
GB9809951D0 (en) | 1998-05-08 | 1998-07-08 | Univ Cambridge Tech | Binding molecules |
WO1999061057A2 (en) | 1998-05-23 | 1999-12-02 | Tanox, Inc. | Molecules targeting cd40 and tumor cells |
DK1100830T3 (da) | 1998-07-28 | 2004-01-19 | Micromet Ag | Heterominiantistoffer |
WO2000018806A1 (de) | 1998-09-25 | 2000-04-06 | Horst Lindhofer | Bispezifische und trispezifische antikörper, die spezifisch mit induzierbaren oberflächenantigenen als operationelle zielstrukturen reagieren |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US7183387B1 (en) | 1999-01-15 | 2007-02-27 | Genentech, Inc. | Polypeptide variants with altered effector function |
EP2364997A3 (en) | 1999-01-15 | 2012-07-04 | Genentech, Inc. | Polypeptide variants with altered effector function |
IL145335A0 (en) | 1999-03-11 | 2002-06-30 | Micromet Ag | Antibody and chemokine constructs and their use in the treatment of autoimmune diseases |
AU2001249835A1 (en) | 2000-04-03 | 2001-10-15 | Oxford Glycosciences (Uk) Ltd. | Diagnosis and treatment of alzheimer's disease |
JP2004511430A (ja) | 2000-05-24 | 2004-04-15 | イムクローン システムズ インコーポレイティド | 二重特異性免疫グロブリン様抗原結合蛋白および製造方法 |
DE10034607A1 (de) | 2000-07-20 | 2002-02-07 | Gundram Jung | Multispezifisches Reagenz zur selektiven Stimulierung von Zelloberflächenrezeptoren |
AU2002212225A1 (en) | 2000-09-08 | 2002-03-22 | Micromet Ag | Antibody and/or chemokine constructs which bind to a chemokine receptor and their use in immunological disorders |
ES2727425T3 (es) | 2000-12-12 | 2019-10-16 | Medimmune Llc | Moléculas con semividas prolongadas, composiciones y usos de las mismas |
CN1294148C (zh) | 2001-04-11 | 2007-01-10 | 中国科学院遗传与发育生物学研究所 | 环状单链三特异抗体 |
JP2005532253A (ja) | 2001-10-25 | 2005-10-27 | ジェネンテック・インコーポレーテッド | 糖タンパク質組成物 |
US20030190705A1 (en) | 2001-10-29 | 2003-10-09 | Sunol Molecular Corporation | Method of humanizing immune system molecules |
US20040002587A1 (en) | 2002-02-20 | 2004-01-01 | Watkins Jeffry D. | Fc region variants |
WO2003107218A1 (ja) | 2002-05-31 | 2003-12-24 | セレスター・レキシコ・サイエンシズ株式会社 | 相互作用予測装置 |
SI1517921T1 (sl) * | 2002-06-28 | 2006-10-31 | Domantis Ltd | Dvojno-specificni ligandi z zvisano serumsko razpolovno dobo |
US20060235208A1 (en) | 2002-09-27 | 2006-10-19 | Xencor, Inc. | Fc variants with optimized properties |
GB0224082D0 (en) | 2002-10-16 | 2002-11-27 | Celltech R&D Ltd | Biological products |
KR20100050587A (ko) | 2002-10-17 | 2010-05-13 | 젠맵 에이/에스 | Cd20에 대한 인간 모노클로날 항체 |
JP4532409B2 (ja) | 2003-01-23 | 2010-08-25 | 小野薬品工業株式会社 | ヒトpd−1に対し特異性を有する物質 |
US8388955B2 (en) | 2003-03-03 | 2013-03-05 | Xencor, Inc. | Fc variants |
JP2004321100A (ja) | 2003-04-25 | 2004-11-18 | Rikogaku Shinkokai | IgGのFc領域を含むタンパク質の変異体 |
UA101945C2 (uk) | 2003-05-30 | 2013-05-27 | Дженентек, Инк. | Лікування злоякісного новоутворення за допомогою бевацизумабу |
WO2004106375A1 (en) | 2003-05-30 | 2004-12-09 | Merus Biopharmaceuticals B.V. I.O. | Fab library for the preparation of anti vegf and anti rabies virus fabs |
US7288638B2 (en) | 2003-10-10 | 2007-10-30 | Bristol-Myers Squibb Company | Fully human antibodies against human 4-1BB |
WO2005063815A2 (en) | 2003-11-12 | 2005-07-14 | Biogen Idec Ma Inc. | Fcϝ receptor-binding polypeptide variants and methods related thereto |
KR20130133302A (ko) | 2003-12-10 | 2013-12-06 | 메다렉스, 인코포레이티드 | Ip―10 항체 및 그의 용도 |
US7235641B2 (en) | 2003-12-22 | 2007-06-26 | Micromet Ag | Bispecific antibodies |
KR20060132006A (ko) | 2004-03-23 | 2006-12-20 | 비오겐 아이덱 엠에이 아이엔씨. | 수용체 커플링제 및 이의 치료적 용도 |
GB0409799D0 (en) | 2004-04-30 | 2004-06-09 | Isis Innovation | Method of generating improved immune response |
ES2526343T3 (es) | 2004-06-03 | 2015-01-09 | Novimmune Sa | Anticuerpos anti-CD3 y métodos de uso de los mismos |
CN1984931B (zh) * | 2004-06-03 | 2012-11-28 | 诺维莫尼公司 | 抗-cd3抗体及其使用方法 |
DK1776384T3 (da) | 2004-08-04 | 2013-09-02 | Mentrik Biotech Llc | VARIANT-Fc-REGIONER |
WO2006028936A2 (en) | 2004-09-02 | 2006-03-16 | Genentech, Inc. | Heteromultimeric molecules |
US7632497B2 (en) | 2004-11-10 | 2009-12-15 | Macrogenics, Inc. | Engineering Fc Antibody regions to confer effector function |
US8728828B2 (en) | 2004-12-22 | 2014-05-20 | Ge Healthcare Bio-Sciences Ab | Purification of immunoglobulins |
CA2602663A1 (en) | 2005-03-31 | 2006-10-05 | Xencor, Inc. | Fc variants with optimized properties |
CA2957144C (en) | 2005-04-08 | 2020-06-02 | Chugai Seiyaku Kabushiki Kaisha | Antibody substituting for function of blood coagulation factor viii |
KR101259655B1 (ko) | 2005-04-15 | 2013-04-30 | 제넨테크, 인크. | Hgf 베타 사슬 변이체 |
JP5085322B2 (ja) | 2005-06-10 | 2012-11-28 | 中外製薬株式会社 | sc(Fv)2を含有する医薬組成物 |
WO2007002543A2 (en) | 2005-06-23 | 2007-01-04 | Medimmune, Inc. | Antibody formulations having optimized aggregation and fragmentation profiles |
JP2009514537A (ja) * | 2005-11-03 | 2009-04-09 | ジェネンテック・インコーポレーテッド | 治療的抗her2抗体融合ポリペプチド |
EP1820513A1 (en) | 2006-02-15 | 2007-08-22 | Trion Pharma Gmbh | Destruction of tumor cells expressing low to medium levels of tumor associated target antigens by trifunctional bispecific antibodies |
TW200745163A (en) | 2006-02-17 | 2007-12-16 | Syntonix Pharmaceuticals Inc | Peptides that block the binding of IgG to FcRn |
AU2007229698B9 (en) | 2006-03-24 | 2012-11-08 | Merck Patent Gmbh | Engineered heterodimeric protein domains |
EP2009101B1 (en) | 2006-03-31 | 2017-10-25 | Chugai Seiyaku Kabushiki Kaisha | Antibody modification method for purifying bispecific antibody |
AU2007258694B2 (en) | 2006-06-06 | 2011-12-22 | Glaxo Group Limited | Administration of anti-CD3 antibodies in the treatment of autoimmune diseases |
US20090182127A1 (en) | 2006-06-22 | 2009-07-16 | Novo Nordisk A/S | Production of Bispecific Antibodies |
WO2008010991A2 (en) | 2006-07-17 | 2008-01-24 | Quintessence Biosciences, Inc. | Methods and compositions for the treatment of cancer |
CA2673470A1 (en) * | 2006-12-21 | 2008-07-03 | Macrogenics, Inc. | Methods for the treatment of lada and other adult-onset autoimmune diabetes using immunosuppressive monoclonal antibodies with reduced toxicity |
KR101540822B1 (ko) | 2007-03-27 | 2015-07-30 | 씨 레인 바이오테크놀로지스, 엘엘씨 | 항체 대용물 경쇄 서열을 포함하는 구축물 및 라이브러리 |
AU2008270710A1 (en) | 2007-06-29 | 2009-01-08 | Merck Sharp & Dohme Corp. | MDL-1 uses |
KR101570252B1 (ko) | 2007-07-17 | 2015-11-19 | 메다렉스, 엘.엘.시. | 글리피칸-3에 대한 단클론 항체 |
JP5334319B2 (ja) | 2007-09-26 | 2013-11-06 | 中外製薬株式会社 | Cdrのアミノ酸置換により抗体の等電点を改変する方法 |
EP2203180B1 (en) * | 2007-10-22 | 2012-11-21 | Merck Serono S.A. | Single ifn-beta fused to a mutated igg fc fragment |
US8242247B2 (en) | 2007-12-21 | 2012-08-14 | Hoffmann-La Roche Inc. | Bivalent, bispecific antibodies |
US20090162359A1 (en) | 2007-12-21 | 2009-06-25 | Christian Klein | Bivalent, bispecific antibodies |
CA2709847C (en) | 2008-01-07 | 2018-07-10 | Amgen Inc. | Method for making antibody fc-heterodimeric molecules using electrostatic steering effects |
JP6018361B2 (ja) | 2008-01-31 | 2016-11-02 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | 制御性t細胞活性を抑制するための、ヒトcd39に対する抗体およびその使用 |
WO2009100140A1 (en) | 2008-02-04 | 2009-08-13 | Medarex, Inc. | Anti-clta-4 antibodies with reduced blocking of binding of ctla-4 to b7 and uses thereof |
NZ588554A (en) * | 2008-04-29 | 2013-03-28 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
AR072999A1 (es) | 2008-08-11 | 2010-10-06 | Medarex Inc | Anticuerpos humanos que se unen al gen 3 de activacion linfocitaria (lag-3) y los usos de estos |
RU2547600C2 (ru) | 2008-10-01 | 2015-04-10 | Эмджен Рисерч (Мьюник) Гмбх | Pscaxcd3, cd19xcd3, c-metxcd3, эндосиалинxcd3, epcamxcd3, igf-1rxcd3 или fap-альфаxcd3 биспецифическое одноцепочечное антитело с межвидовой специфичностью |
NZ592611A (en) | 2008-10-10 | 2013-01-25 | Emergent Product Dev Seattle | A single chain fusion protein that binds to tcr complex and does not induce or induces a minimally detectable cytokine release |
DE202008016028U1 (de) | 2008-12-04 | 2010-04-15 | Melitta Haushaltsprodukte Gmbh & Co. Kg | Behälter zur Aufbewahrung von Gegenständen |
UA109633C2 (uk) * | 2008-12-09 | 2015-09-25 | Антитіло людини проти тканинного фактора | |
AU2009335798B2 (en) * | 2008-12-19 | 2014-11-27 | Macrogenics, Inc. | Covalent diabodies and uses thereof |
US20120149875A1 (en) | 2009-01-12 | 2012-06-14 | Ge Healthcare Bio-Sciences Ab | Affinity chromatography matrix |
AU2010206681A1 (en) | 2009-01-23 | 2011-09-01 | Biogen Idec Ma Inc. | Stabilized Fc polypeptides with reduced effector function and methods of use |
JP5836807B2 (ja) | 2009-03-05 | 2015-12-24 | アッヴィ・インコーポレイテッド | Il−17結合タンパク質 |
WO2010120561A1 (en) | 2009-04-01 | 2010-10-21 | Genentech, Inc. | Anti-fcrh5 antibodies and immunoconjugates and methods of use |
JP2012525149A (ja) | 2009-04-27 | 2012-10-22 | オンコメッド ファーマシューティカルズ インコーポレイテッド | ヘテロ多量体分子を作製するための方法 |
JP5375323B2 (ja) | 2009-05-15 | 2013-12-25 | 株式会社ニコン | 移動体装置、露光装置、及び移動体制御方法 |
BRPI1007602A2 (pt) | 2009-05-27 | 2016-02-16 | Hoffmann La Roche | "anticorpo tri ou tetraespecífico, método para preparação de um anticorpo triespecífico ou tetraespecífico, célula hospedeira, composição, composição farmacêutica e método para o tratamento de um paciente com necessidade de terapia" |
CN102471378B (zh) | 2009-06-26 | 2014-04-02 | 瑞泽恩制药公司 | 容易地分离的具有天然免疫球蛋白形式的双特异性抗体 |
CN105131112A (zh) | 2009-08-29 | 2015-12-09 | Abbvie公司 | 治疗用dll4结合蛋白 |
PT2483310E (pt) | 2009-09-29 | 2014-10-07 | Roche Glycart Ag | Anticorpos bi-específicos agonistas do receptor de morte |
CA2778334A1 (en) | 2009-10-20 | 2011-04-28 | Charlotte Mckee | Anti-cd3 antibody dosing in autoimmune disease |
US10053513B2 (en) | 2009-11-30 | 2018-08-21 | Janssen Biotech, Inc. | Antibody Fc mutants with ablated effector functions |
KR102380315B1 (ko) | 2009-12-25 | 2022-03-29 | 추가이 세이야쿠 가부시키가이샤 | 폴리펩티드 다량체를 정제하기 위한 폴리펩티드의 개변방법 |
US20120021409A1 (en) | 2010-02-08 | 2012-01-26 | Regeneron Pharmaceuticals, Inc. | Common Light Chain Mouse |
US10435458B2 (en) | 2010-03-04 | 2019-10-08 | Chugai Seiyaku Kabushiki Kaisha | Antibody constant region variants with reduced Fcgammar binding |
TWI653333B (zh) | 2010-04-01 | 2019-03-11 | 安進研究(慕尼黑)有限責任公司 | 跨物種專一性之PSMAxCD3雙專一性單鏈抗體 |
EP3539988A3 (en) | 2010-05-27 | 2019-12-04 | Genmab A/S | Monoclonal antibodies against her2 |
DK2644698T3 (en) * | 2010-11-17 | 2018-01-22 | Chugai Pharmaceutical Co Ltd | MULTI-SPECIFIC ANTIGEN-BINDING MOLECULE WITH ALTERNATIVE FUNCTION TO BLOOD COAGULATION FACTOR FUNCTION VIII |
TR201815863T4 (tr) | 2010-11-30 | 2018-11-21 | Chugai Pharmaceutical Co Ltd | Sitotoksisiteyi indükleyen terapötik madde. |
US20120195900A1 (en) * | 2010-12-22 | 2012-08-02 | Abbott Laboratories | Tri-variable domain binding proteins and uses thereof |
DK2663580T3 (en) | 2011-01-10 | 2017-03-13 | Ct Atlantic Ltd | COMBINATION THERAPY INCLUDING TUMOR ASSOCIATED ANTI-BINDING ANTIBODIES |
EP2673297A2 (en) | 2011-02-11 | 2013-12-18 | Zyngenia, Inc. | Monovalent and multivalent multispecific complexes and uses thereof |
MX352889B (es) | 2011-02-25 | 2017-12-13 | Chugai Pharmaceutical Co Ltd | Anticuerpo de fc especifico para fcyriib. |
MX358752B (es) | 2011-03-25 | 2018-08-31 | Glenmark Pharmaceuticals Sa | Inmunoglobulinas heterodiméricas. |
AU2012233313C1 (en) | 2011-03-30 | 2017-08-03 | Chugai Seiyaku Kabushiki Kaisha | Method for altering plasma retention and immunogenicity of antigen-binding molecule |
TR201905909T4 (tr) | 2011-04-19 | 2019-05-21 | Pfizer | Kanser tedavisi için anti-4-1bb antikorlarının ve adcc indükleyici antikorların kombinasyonları. |
EP2537864B1 (en) | 2011-06-24 | 2019-08-07 | Laboratoire Français du Fractionnement et des Biotechnologies | Fc variants with reduced effector functions |
KR20140058532A (ko) | 2011-06-30 | 2014-05-14 | 겐자임 코포레이션 | T-세포 활성화 억제제 |
US8790651B2 (en) * | 2011-07-21 | 2014-07-29 | Zoetis Llc | Interleukin-31 monoclonal antibody |
HUE038225T2 (hu) | 2011-08-23 | 2018-10-29 | Roche Glycart Ag | Bispecifikus, T-sejtaktiváló antigénkötõ, molekulák |
PT2748202T (pt) | 2011-08-23 | 2018-10-08 | Roche Glycart Ag | Moléculas de ligação ao antigénio biespecíficas |
ES2732712T3 (es) * | 2011-10-31 | 2019-11-25 | Chugai Pharmaceutical Co Ltd | Molécula de unión a antígeno que tiene una conjugación regulada entre la cadena pesada y la cadena ligera |
WO2013070468A1 (en) | 2011-11-08 | 2013-05-16 | The Trustees Of The University Of Pennsylvania | Glypican-3-specific antibody and uses thereof |
EP2780372B1 (en) | 2011-11-17 | 2019-01-02 | Jung, Gundram | Bi-specific antibodies for medical use |
EA201400709A1 (ru) | 2011-12-19 | 2016-08-31 | Синиммун Гмбх | Молекула биспецифического антитела |
WO2013116662A1 (en) | 2012-02-03 | 2013-08-08 | Interdigital Patent Holdings, Inc. | Method and apparatus for coexistence among wireless transmit/receive units (wtrus) operating in the same spectrum |
PT2825559T (pt) | 2012-03-13 | 2019-06-07 | Novimmune Sa | Anticorpos biespecíficos prontamente isolados com formato de imunoglobulina nativo |
RU2673153C2 (ru) | 2012-04-20 | 2018-11-22 | АПТЕВО РИСЁРЧ ЭНД ДИВЕЛОПМЕНТ ЭлЭлСи | Полипептиды, связывающиеся с cd3 |
US20140154270A1 (en) | 2012-05-21 | 2014-06-05 | Chen Wang | Purification of non-human antibodies using kosmotropic salt enhanced protein a affinity chromatography |
WO2013181543A1 (en) | 2012-06-01 | 2013-12-05 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | High-affinity monoclonal antibodies to glypican-3 and use thereof |
SG10201605703TA (en) | 2012-07-06 | 2016-09-29 | Genmab Bv | Dimeric protein with triple mutations |
WO2014028560A2 (en) | 2012-08-14 | 2014-02-20 | Ibc Pharmaceuticals, Inc. | T-cell redirecting bispecific antibodies for treatment of disease |
JOP20200236A1 (ar) | 2012-09-21 | 2017-06-16 | Regeneron Pharma | الأجسام المضادة لمضاد cd3 وجزيئات ربط الأنتيجين ثنائية التحديد التي تربط cd3 وcd20 واستخداماتها |
SG10201705787VA (en) | 2012-09-27 | 2017-08-30 | Merus Nv | BISPECIFIC IgG ANTIBODIES AS T CELL ENGAGERS |
CN103833852A (zh) | 2012-11-23 | 2014-06-04 | 上海市肿瘤研究所 | 针对磷脂酰肌醇蛋白多糖-3和t细胞抗原的双特异性抗体 |
EP3508215A3 (en) | 2012-12-03 | 2019-10-02 | Bristol-Myers Squibb Company | Enhancing anti-cancer activity of immunomodulatory fc fusion proteins |
WO2014116846A2 (en) | 2013-01-23 | 2014-07-31 | Abbvie, Inc. | Methods and compositions for modulating an immune response |
AU2014225788B2 (en) | 2013-03-05 | 2018-03-29 | Baylor College Of Medicine | Engager cells for immunotherapy |
EP2970486B1 (en) | 2013-03-15 | 2018-05-16 | Xencor, Inc. | Modulation of t cells with bispecific antibodies and fc fusions |
ES2699599T3 (es) | 2013-03-15 | 2019-02-11 | Abbvie Biotherapeutics Inc | Variantes de Fc |
WO2014165818A2 (en) | 2013-04-05 | 2014-10-09 | T Cell Therapeutics, Inc. | Compositions and methods for preventing and treating prostate cancer |
EP3016681B1 (en) | 2013-07-05 | 2019-12-18 | Genmab A/S | Humanized or chimeric cd3 antibodies |
JP6534615B2 (ja) | 2013-09-27 | 2019-06-26 | 中外製薬株式会社 | ポリペプチド異種多量体の製造方法 |
CA2922950A1 (en) | 2013-09-30 | 2015-04-02 | Shinya Ishii | Method for producing antigen-binding molecule using modified helper phage |
EP3176185A1 (en) | 2013-11-04 | 2017-06-07 | Glenmark Pharmaceuticals S.A. | Production of t cell retargeting hetero-dimeric immunoglobulins |
MX2016007958A (es) | 2013-12-17 | 2016-08-03 | Genentech Inc | Anticuerpos anti-cd3 y metodos de uso. |
CA2939711C (en) | 2014-02-21 | 2020-09-29 | Cellectis | Method for in situ inhibition of regulatory t cells |
RS59907B1 (sr) | 2014-03-28 | 2020-03-31 | Xencor Inc | Bispecifična antitela koja se vezuju za cd38 i cd3 |
AU2015244814B2 (en) | 2014-04-07 | 2020-12-24 | Chugai Seiyaku Kabushiki Kaisha | Immunoactivating antigen-binding molecule |
WO2015164392A2 (en) | 2014-04-21 | 2015-10-29 | Stemcentrx, Inc. | Novel antii-rnf43 antibodies and methods of use |
AU2015260230A1 (en) | 2014-05-13 | 2016-11-17 | Chugai Seiyaku Kabushiki Kaisha | T cell-redirected antigen-binding molecule for cells having immunosuppression function |
KR20160002132A (ko) | 2014-06-30 | 2016-01-07 | 삼성전자주식회사 | 음장 효과를 제공하기 위한 전자 장치 및 방법 |
AR101262A1 (es) | 2014-07-26 | 2016-12-07 | Regeneron Pharma | Plataforma de purificación para anticuerpos biespecíficos |
CN105444789A (zh) | 2014-08-25 | 2016-03-30 | 同方威视技术股份有限公司 | 一种光纤光栅解调仪及其温度控制方法 |
MA40764A (fr) | 2014-09-26 | 2017-08-01 | Chugai Pharmaceutical Co Ltd | Agent thérapeutique induisant une cytotoxicité |
WO2016179003A1 (en) | 2015-05-01 | 2016-11-10 | Genentech, Inc. | Masked anti-cd3 antibodies and methods of use |
EP3305322A4 (en) | 2015-06-05 | 2018-12-26 | Chugai Seiyaku Kabushiki Kaisha | Combined use of immune activators |
EP3320773B9 (en) | 2015-07-10 | 2023-10-11 | Chugai Seiyaku Kabushiki Kaisha | Mouse having human cd3 gene substituted for endogenous cd3 gene |
US11649293B2 (en) | 2015-11-18 | 2023-05-16 | Chugai Seiyaku Kabushiki Kaisha | Method for enhancing humoral immune response |
JP6931329B2 (ja) | 2015-11-18 | 2021-09-01 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子を用いた併用療法 |
EP3398965A4 (en) | 2015-12-28 | 2019-09-18 | Chugai Seiyaku Kabushiki Kaisha | METHOD FOR PROMOTING THE EFFICACY OF PURIFYING A POLYPEPTIDE CONTAINING AN FC REGION |
AU2017233658B2 (en) | 2016-03-14 | 2023-09-21 | Chugai Seiyaku Kabushiki Kaisha | Cell injury inducing therapeutic drug for use in cancer therapy |
US20200123256A1 (en) * | 2017-05-02 | 2020-04-23 | Chugai Seiyaku Kabushiki Kaisha | Cytotoxicity-inducing therapeutic agent |
AU2018338859A1 (en) * | 2017-09-29 | 2020-02-06 | Chugai Seiyaku Kabushiki Kaisha | Multispecific antigen-binding molecule having blood coagulation factor VIII (FVIII) cofactor function-substituting activity, and pharmaceutical formulation containing said molecule as active ingredient |
TWI817974B (zh) | 2017-12-28 | 2023-10-11 | 日商中外製藥股份有限公司 | 細胞毒性誘導治療劑 |
EP3793599A1 (en) | 2018-05-16 | 2021-03-24 | Janssen Biotech, Inc. | Bcma/cd3 and gprdc5d/cd3 bispecific antibodies for use in cancer therapy |
-
2011
- 2011-11-30 TR TR2018/15863T patent/TR201815863T4/tr unknown
- 2011-11-30 KR KR1020137016886A patent/KR101960109B1/ko active IP Right Grant
- 2011-11-30 ES ES11845786.0T patent/ES2693232T3/es active Active
- 2011-11-30 KR KR1020197006956A patent/KR102244173B1/ko active IP Right Grant
- 2011-11-30 TW TW109134405A patent/TWI736437B/zh active
- 2011-11-30 PL PL11845786T patent/PL2647707T3/pl unknown
- 2011-11-30 WO PCT/JP2011/077603 patent/WO2012073985A1/ja active Application Filing
- 2011-11-30 BR BR112013013311A patent/BR112013013311A2/pt not_active Application Discontinuation
- 2011-11-30 EP EP23208881.5A patent/EP4303236A3/en active Pending
- 2011-11-30 EP EP18192844.1A patent/EP3434767A1/en active Pending
- 2011-11-30 DK DK11845786.0T patent/DK2647707T3/en active
- 2011-11-30 TW TW112104521A patent/TW202323302A/zh unknown
- 2011-11-30 KR KR1020227018833A patent/KR20220082104A/ko not_active Application Discontinuation
- 2011-11-30 TW TW110125458A patent/TWI807362B/zh active
- 2011-11-30 PT PT11845786T patent/PT2647707T/pt unknown
- 2011-11-30 CA CA2819530A patent/CA2819530C/en active Active
- 2011-11-30 SI SI201131599T patent/SI2647707T1/sl unknown
- 2011-11-30 TW TW100143903A patent/TWI638833B/zh active
- 2011-11-30 RU RU2013129694A patent/RU2663123C2/ru active
- 2011-11-30 US US13/990,088 patent/US11066483B2/en active Active
- 2011-11-30 KR KR1020247005207A patent/KR20240025059A/ko active Application Filing
- 2011-11-30 EP EP23208888.0A patent/EP4303237A3/en active Pending
- 2011-11-30 SG SG2013041587A patent/SG190726A1/en unknown
- 2011-11-30 JP JP2012546900A patent/JP6278598B2/ja active Active
- 2011-11-30 KR KR1020217009395A patent/KR102447595B1/ko active IP Right Grant
- 2011-11-30 LT LTEP11845786.0T patent/LT2647707T/lt unknown
- 2011-11-30 TW TW108126743A patent/TWI709574B/zh active
- 2011-11-30 CN CN201811065101.1A patent/CN109160951A/zh active Pending
- 2011-11-30 SG SG10201506840QA patent/SG10201506840QA/en unknown
- 2011-11-30 EP EP23200977.9A patent/EP4279513A3/en active Pending
- 2011-11-30 MX MX2013006100A patent/MX349057B/es active IP Right Grant
- 2011-11-30 CN CN201180068471.0A patent/CN103429737B/zh active Active
- 2011-11-30 EP EP23200956.3A patent/EP4279511A3/en active Pending
- 2011-11-30 EP EP11845786.0A patent/EP2647707B1/en not_active Revoked
- 2011-11-30 AR ARP110104461A patent/AR084053A1/es unknown
- 2011-11-30 EP EP23200969.6A patent/EP4279512A3/en active Pending
- 2011-11-30 TW TW105144146A patent/TWI685503B/zh active
- 2011-11-30 AU AU2011337697A patent/AU2011337697B2/en active Active
-
2014
- 2014-02-07 HK HK19101521.5A patent/HK1259128A1/zh unknown
-
2017
- 2017-01-27 AU AU2017200565A patent/AU2017200565A1/en not_active Abandoned
- 2017-02-14 AU AU2017200999A patent/AU2017200999B2/en active Active
-
2018
- 2018-01-16 JP JP2018004616A patent/JP6719488B2/ja active Active
- 2018-10-05 HR HRP20181615TT patent/HRP20181615T1/hr unknown
-
2020
- 2020-02-25 JP JP2020029153A patent/JP6773929B2/ja active Active
- 2020-10-01 JP JP2020166667A patent/JP6963665B2/ja active Active
-
2021
- 2021-07-06 US US17/367,909 patent/US20220041756A1/en active Pending
- 2021-10-15 JP JP2021169248A patent/JP2022023133A/ja not_active Withdrawn
-
2023
- 2023-06-30 JP JP2023107618A patent/JP2023115329A/ja active Pending
Patent Citations (35)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3773719A (en) | 1966-12-06 | 1973-11-20 | Hoffmann La Roche | 2-aminoxy-2'-acyl-acetanilide |
EP0058481A1 (en) | 1981-02-16 | 1982-08-25 | Zeneca Limited | Continuous release pharmaceutical compositions |
EP0133988A2 (de) | 1983-08-02 | 1985-03-13 | Hoechst Aktiengesellschaft | Regulatorische Peptide enthaltende pharmazeutische Präparate mit protrahierter Freisetzung und Verfahren zu deren Herstellung |
EP0239400A2 (en) | 1986-03-27 | 1987-09-30 | Medical Research Council | Recombinant antibodies and methods for their production |
WO1988001649A1 (en) | 1986-09-02 | 1988-03-10 | Genex Corporation | Single polypeptide chain binding molecules |
US5260203A (en) | 1986-09-02 | 1993-11-09 | Enzon, Inc. | Single polypeptide chain binding molecules |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
WO1992020791A1 (en) | 1990-07-10 | 1992-11-26 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1992001047A1 (en) | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1992003918A1 (en) | 1990-08-29 | 1992-03-19 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO1993006213A1 (en) | 1991-09-23 | 1993-04-01 | Medical Research Council | Production of chimeric antibodies - a combinatorial approach |
WO1993011236A1 (en) | 1991-12-02 | 1993-06-10 | Medical Research Council | Production of anti-self antibodies from antibody segment repertoires and displayed on phage |
WO1993012227A1 (en) | 1991-12-17 | 1993-06-24 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO1993019172A1 (en) | 1992-03-24 | 1993-09-30 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1994002602A1 (en) | 1992-07-24 | 1994-02-03 | Cell Genesys, Inc. | Generation of xenogeneic antibodies |
WO1994011523A2 (en) | 1992-11-13 | 1994-05-26 | Idec Pharmaceuticals Corporation | Fully impaired consensus kozac sequences for mammalian expression |
WO1994025585A1 (en) | 1993-04-26 | 1994-11-10 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO1995001438A1 (en) | 1993-06-30 | 1995-01-12 | Medical Research Council | Sbp members with a chemical moiety covalently bound within the binding site; production and selection thereof |
WO1995015388A1 (en) | 1993-12-03 | 1995-06-08 | Medical Research Council | Recombinant binding proteins and peptides |
WO1996002576A1 (fr) | 1994-07-13 | 1996-02-01 | Chugai Seiyaku Kabushiki Kaisha | Anticorps humain reconstitue contre l'interleukine-8 humaine |
WO1996027011A1 (en) | 1995-03-01 | 1996-09-06 | Genentech, Inc. | A method for making heteromultimeric polypeptides |
WO1996033735A1 (en) | 1995-04-27 | 1996-10-31 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
WO1996034096A1 (en) | 1995-04-28 | 1996-10-31 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
WO2003000883A1 (en) | 2001-06-22 | 2003-01-03 | Chugai Seiyaku Kabushiki Kaisha | Cell proliferation inhibitors containing anti-glypican 3 antibody |
WO2003104453A1 (ja) | 2002-06-05 | 2003-12-18 | 中外製薬株式会社 | 抗体作製方法 |
WO2004022754A1 (ja) | 2002-09-04 | 2004-03-18 | Chugai Seiyaku Kabushiki Kaisha | MRL/lprマウスを用いた抗体の作製 |
WO2004065611A1 (ja) | 2003-01-21 | 2004-08-05 | Chugai Seiyaku Kabushiki Kaisha | 抗体の軽鎖スクリーニング方法 |
WO2006006693A1 (ja) | 2004-07-09 | 2006-01-19 | Chugai Seiyaku Kabushiki Kaisha | 抗グリピカン3抗体 |
WO2006106905A1 (ja) | 2005-03-31 | 2006-10-12 | Chugai Seiyaku Kabushiki Kaisha | 会合制御によるポリペプチド製造方法 |
WO2006132352A1 (ja) | 2005-06-10 | 2006-12-14 | Chugai Seiyaku Kabushiki Kaisha | sc(Fv)2を含有する医薬組成物 |
WO2008090960A1 (ja) * | 2007-01-24 | 2008-07-31 | Kyowa Hakko Kirin Co., Ltd. | ガングリオシドgm2に特異的に結合する遺伝子組換え抗体組成物 |
WO2009011941A2 (en) | 2007-04-04 | 2009-01-22 | The Government Of U.S.A., As Represented By The Secretary, Departmetnt Of Health & Human Services | Monoclonal antibodies against dengue and other viruses with deletion in fc region |
WO2009041613A1 (ja) * | 2007-09-26 | 2009-04-02 | Chugai Seiyaku Kabushiki Kaisha | 抗体定常領域改変体 |
WO2009120922A2 (en) * | 2008-03-27 | 2009-10-01 | Zymogenetics, Inc. | Compositions and methods for inhibiting pdgfrbeta and vegf-a |
WO2010034441A1 (en) * | 2008-09-26 | 2010-04-01 | F. Hoffmann-La Roche Ag | Bispecific anti-egfr/anti-igf-1r antibodies |
Non-Patent Citations (90)
Title |
---|
"Current protocols in Immunology", 1993, JOHN WILEY & SONS, INC. |
"Epitope Mapping Protocols in Methods in Molecular Biology", vol. 66, 1996 |
"New Comprehensive Biochemistry", vol. 18B, 1988, ELSEVIER SCIENCE PUBLISHERS BV., article "Hormones and their Actions Part II", pages: 1 - 46 |
"Remington's Pharmaceutical Science", 1980 |
"Remington's Pharmaceutical Science", MARK PUBLISHING COMPANY |
"SAIBO KOGAKU", 1994, SHUJUNSHA, article "Secchaku Inshi" |
"Sequences of proteins of immunological interest", NIH PUBLICATION NO.91-3242 |
AMANN M ET AL.: "Therapeutic window of an EpCAM/ CD3-specific BiTE antibody in mice is determined by a subpopulation of EpCAM- expressing lymphocytes that is absent in humans", CANCER IMMUNOL. IMMUNOTHER., vol. 58, no. 1, 2009, pages 95 - 109, XP019654574 * |
ANAL. BIOCHEM., vol. 162, no. 1, 1987, pages 156 - 159 |
BIOCHEMISTRY, vol. 18, no. 24, 1979, pages 5294 - 5299 |
BIOPOLYMERS, vol. 22, 1983, pages 547 - 556 |
BLOOD, vol. 109, 2007, pages 1185 - 1192 |
BLOOD, vol. 76, no. 1, 1990, pages 31 - 35 |
C. EUR. J. IMMUNOL., vol. 6, no. 7, 1976, pages 511 - 519 |
CANCER IMMUNOL IMMUNOTHER, vol. 55, no. 5, 2006, pages 503 - 14 |
CANCER IMMUNOL IMMUNOTHER, vol. 56, no. 10, 2007, pages 1637 - 44 |
CANCER IMMUNOL IMMUNOTHER, vol. 58, no. 1, 2009, pages 95 - 109 |
CANCER SCI., vol. 101, no. 10, 2010, pages 2227 - 33 |
CANCER TREAT REV., vol. 36, no. 6, 2010, pages 458 - 67 |
CELL, vol. 57, no. 2, 1989, pages 277 - 285 |
CELL, vol. 60, no. 2, 1990, pages 225 - 234 |
CELL, vol. 61, no. 2, 1990, pages 341 - 350 |
CELL, vol. 77, no. 3, 1994, pages 391 - 400 |
CELL, vol. 8, no. 3, 1976, pages 405 - 415 |
CHEMTECH., vol. 12, 1982, pages 98 - 105 |
CLIN CANCER RES., vol. 16, no. 1, 2010, pages 11 - 20 |
CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, vol. 81, 1978, pages 1 - 7 |
DAVIS ET AL., IMMUNOLOGICAL REVIEWS, vol. 190, 2002, pages 123 - 136 |
DRUG DES DEVEL THER, vol. 3, 2009, pages 7 - 16 |
DRUG DISCOV TODAY, vol. 10, no. 18, 2005, pages 1237 - 44 |
EBERT, K. M. ET AL., BIO/TECHNOLOGY, vol. 12, no. 7, 1994, pages 699 - 702 |
ED HARLOW; DAVID LANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY, pages: 359 - 420 |
EMBO J., vol. 12, no. 7, 1993, pages 2645 - 53 |
EXPERT OPIN BIOL THER, vol. 10, no. 8, 2010, pages 1259 - 69 |
FANTL ET AL., ANNU. REV. BIOCHEM., vol. 62, 1993, pages 453 - 481 |
FLOWER DR, BIOCHIM. BIOPHYS. ACTA, FLOWER (BIOCHIM. BIOPHYS. ACTA, vol. 1422, no. 3, 1999, pages 207 - 234 |
HUM. ANTIBOD. HYBRIDOMAS, vol. 1, no. 1, 1990, pages 47 - 54 |
INT J CANCER, vol. 103, no. 4, 2003, pages 455 - 65 |
INT J CANCER, vol. 41, no. 4, 1988, pages 609 - 15 |
IZUMI KUMAGAI ET AL.: "Humanized bispecific antibodies retargeting of lymphocytes against tumor cells", DRUG DELIVERY SYSTEM, vol. 23, no. 5, 2008, pages 518 - 525, XP055108472 * |
J IMMUNOL. METHODS, vol. 231, no. 1-2, 1999, pages 177 - 189 |
J. BIOMED. MATER. RES., vol. 15, 1981, pages 267 - 277 |
J. EXP. MED., vol. 148, no. 1, 1978, pages 313 - 323 |
J. IMMUNOL. METHODS, vol. 35, no. 1-2, 1980, pages 1 - 21 |
J. IMMUNOL., vol. 123, no. 4, 1979, pages 1548 - 1550 |
J. MOL. RECOGNIT, vol. 16, 2003, pages 113 - 120 |
J. MOL. RECOGNIT., vol. 16, 2003, pages 113 - 120 |
JOURNAL OF IMMUNOLOGY, vol. 152, no. 11, 1994, pages 5368 - 5374 |
KABAT EA ET AL.: "Sequence of Proteins of Immunological Interest", 1991, NIH |
KOHLER; MILSTEIN ET AL., METHODS ENZYMOL., vol. 73, 1981, pages 3 - 46 |
MASAMI SUZUKI: "Kotai Iyakuhin no Kenkyu Kaihatsu", NISSEIKEN TAYORI, vol. 56, no. 4, 1 July 2010 (2010-07-01), pages 45 - 51, XP008168904 * |
MASSAGUE, CELL, vol. 69, no. 6, 1992, pages 1067 - 1070 |
MCEARCHERN JA ET AL.: "Engineered anti-CD70 antibody with multiple effector functions exhibits in vitro and in vivo antitumor activities", BLOOD, vol. 109, no. 3, 2007, pages 1185 - 1192, XP008123428 * |
MERCHANT AM ET AL., NAT. BIOTECHNOL., vol. 16, 1998, pages 677 - 681 |
MERCHANT AM ET AL., NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 677 - 681 |
MERCHANT AM ET AL.: "An efficient route to human bispecific IgG", NAT. BIOTECHNOL., vol. 16, no. 7, 1998, pages 677 - 681, XP002141015 * |
MILSTEIN C ET AL., NATURE, vol. 305, 1983, pages 537 - 540 |
MIYAJIMA ET AL., ANNU. REV. IMMUNOL., vol. 10, 1992, pages 295 - 331 |
NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 677 - 681 |
NATURE, vol. 276, no. 5685, 1978, pages 269 - 270 |
NATURE, vol. 277, no. 5692, 1979, pages 131 - 133 |
NATURE, vol. 313, no. 6005, 1985, pages 756 - 761 |
NATURE, vol. 314, no. 6012, 1985, pages 628 - 31 |
NUCLEIC ACIDS RES., vol. 17, no. 8, 1989, pages 2919 - 2932 |
PATTHY, CELL, vol. 61, no. 1, 1990, pages 13 - 14 |
PLUCKTHUN: "The Pharmacology of Monoclonal Antibodies", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315 |
PROC NATL ACAD SCI USA, vol. 83, no. 5, 1986, pages 1453 - 7 |
PROC NATL ACAD SCI USA., vol. 92, no. 15, 1995, pages 7021 - 5 |
PROC. NATL. ACAD. SCI. U.S.A., vol. 85, no. 16, 1988, pages 5879 - 5883 |
PROC. NATL. ACAD. SCI. USA, vol. 103, no. 11, 2006, pages 4005 - 4010 |
PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4914 - 4917 |
PROC. NATL. ACAD. SCI. USA, vol. 85, no. 23, 1988, pages 8998 - 9002 |
PROC. NATL. ACAD. SCI. USA, vol. 89, no. 12, 1992, pages 5640 - 5644 |
PROC. NATL. ACAD. SCI. USA, vol. 91, no. 2, 1994, pages 459 - 463 |
PROC. NATL. ACAD. SCI. USA., vol. 85, no. 10, 1988, pages 3435 - 3439 |
PROC. NATL. ACAD. SCI. USA., vol. 86, no. 1, 1989, pages 27 - 31 |
PROC. NATL. ACAD. SCI. USA., vol. 87, no. 22, 1990, pages 8702 - 8706 |
PROTEIN ENGINEERING, vol. 9, no. 3, 1996, pages 299 - 305 |
RIDGWAY JB ET AL., PROTEIN ENGINEERING, vol. 9, 1996, pages 617 - 621 |
SAMBROOK, J ET AL.: "Molecular Cloning", 1989, COLD SPRING HARBOR LAB. PRESS, pages: 9.47 - 9.58 |
SATO, K. ET AL., CANCER RES., vol. 53, 1993, pages 851 - 856 |
SCIENCE, vol. 240, no. 4855, 1988, pages 1038 - 1041 |
SCIENCE, vol. 321, no. 5891, 2008, pages 974 - 7 |
SEBASTIAN M ET AL.: "Treatment of non-small cell lung cancer patients with the trifunctional monoclonal antibody catumaxomab (anti-EpCAM x anti-CD3): a phase I study", CANCER IMMUNOL. IMMUNOTHER., vol. 56, no. 10, 2007, pages 1637 - 1644, XP019539101 * |
SMITH ET AL., CELL, vol. 76, no. 6, 1994, pages 959 - 962 |
TAGA ET AL., FASEB J., vol. 6, 1992, pages 3387 - 3396 |
TEERINEN T ET AL.: "Structure-based stability engineering of the mouse IgG1 Fab fragment by modifying constant domains", J. MOL. BIOL., vol. 361, no. 4, 2006, pages 687 - 697, XP024951313 * |
TRENDS BIOTECHNOL, vol. 22, no. 5, 2004, pages 238 - 44 |
ULLRICH ET AL., CELL, vol. 61, no. 2, 1990, pages 203 - 212 |
VANDAMME ET AL., EUR. J. BIOCHEM., vol. 192, no. 3, 1990, pages 767 - 775 |
Cited By (179)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11168344B2 (en) | 2005-03-31 | 2021-11-09 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
US10011858B2 (en) | 2005-03-31 | 2018-07-03 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
US9670269B2 (en) | 2006-03-31 | 2017-06-06 | Chugai Seiyaku Kabushiki Kaisha | Methods of modifying antibodies for purification of bispecific antibodies |
US10934344B2 (en) | 2006-03-31 | 2021-03-02 | Chugai Seiyaku Kabushiki Kaisha | Methods of modifying antibodies for purification of bispecific antibodies |
US11046784B2 (en) | 2006-03-31 | 2021-06-29 | Chugai Seiyaku Kabushiki Kaisha | Methods for controlling blood pharmacokinetics of antibodies |
US9828429B2 (en) | 2007-09-26 | 2017-11-28 | Chugai Seiyaku Kabushiki Kaisha | Method of modifying isoelectric point of antibody via amino acid substitution in CDR |
US11332533B2 (en) | 2007-09-26 | 2022-05-17 | Chugai Seiyaku Kabushiki Kaisha | Modified antibody constant region |
US11248053B2 (en) | 2007-09-26 | 2022-02-15 | Chugai Seiyaku Kabushiki Kaisha | Method of modifying isoelectric point of antibody via amino acid substitution in CDR |
US10138293B2 (en) | 2007-12-21 | 2018-11-27 | Hoffmann-La Roche, Inc. | Bivalent, bispecific antibodies |
US10927163B2 (en) | 2007-12-21 | 2021-02-23 | Hoffmann-La Roche, Inc. | Bivalent, bispecific antibodies |
US10640555B2 (en) | 2009-06-16 | 2020-05-05 | Hoffmann-La Roche Inc. | Bispecific antigen binding proteins |
US9676845B2 (en) | 2009-06-16 | 2017-06-13 | Hoffmann-La Roche, Inc. | Bispecific antigen binding proteins |
US11673945B2 (en) | 2009-06-16 | 2023-06-13 | Hoffmann-La Roche Inc. | Bispecific antigen binding proteins |
JP2018093879A (ja) * | 2010-11-30 | 2018-06-21 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
US11066483B2 (en) | 2010-11-30 | 2021-07-20 | Chugai Seiyaku Kabushiki Kaisha | Cytotoxicity-inducing therapeutic agent |
US11618790B2 (en) | 2010-12-23 | 2023-04-04 | Hoffmann-La Roche Inc. | Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery |
US9982036B2 (en) | 2011-02-28 | 2018-05-29 | Hoffmann-La Roche Inc. | Dual FC antigen binding proteins |
US10793621B2 (en) | 2011-02-28 | 2020-10-06 | Hoffmann-La Roche Inc. | Nucleic acid encoding dual Fc antigen binding proteins |
US10611825B2 (en) | 2011-02-28 | 2020-04-07 | Hoffmann La-Roche Inc. | Monovalent antigen binding proteins |
JP2014526895A (ja) * | 2011-08-23 | 2014-10-09 | ロシュ グリクアート アーゲー | 二重特異性抗原結合分子 |
US11639397B2 (en) | 2011-08-23 | 2023-05-02 | Roche Glycart Ag | Bispecific antibodies specific for T-cell activating antigens and a tumor antigen and methods of use |
US11851476B2 (en) | 2011-10-31 | 2023-12-26 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule having regulated conjugation between heavy-chain and light-chain |
US11142563B2 (en) | 2012-06-14 | 2021-10-12 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule containing modified Fc region |
JP2018076304A (ja) * | 2012-06-14 | 2018-05-17 | 中外製薬株式会社 | 改変されたFc領域を含む抗原結合分子 |
JP2021059572A (ja) * | 2012-06-14 | 2021-04-15 | 中外製薬株式会社 | 改変されたFc領域を含む抗原結合分子 |
JP7245815B2 (ja) | 2012-06-14 | 2023-03-24 | 中外製薬株式会社 | 改変されたFc領域を含む抗原結合分子 |
US10106612B2 (en) | 2012-06-27 | 2018-10-23 | Hoffmann-La Roche Inc. | Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof |
US11421022B2 (en) | 2012-06-27 | 2022-08-23 | Hoffmann-La Roche Inc. | Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof |
US11072656B2 (en) | 2012-09-21 | 2021-07-27 | Regeneran Pharmaceuticals, Inc. | Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof |
US11155621B2 (en) | 2012-09-21 | 2021-10-26 | Regeneran Pharmaceuticals, Inc. | Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof |
US9771573B2 (en) | 2012-10-03 | 2017-09-26 | Zymeworks Inc. | Methods of quantitating heavy and light chain polypeptide pairs |
US10323239B2 (en) | 2012-10-04 | 2019-06-18 | Research Development Foundation | Serine protease molecules and therapies |
JP2017140051A (ja) * | 2012-10-04 | 2017-08-17 | リサーチ ディベロップメント ファウンデーション | セリンプロテアーゼ分子および療法 |
US11535838B2 (en) | 2012-10-04 | 2022-12-27 | Research Development Foundation | Serine protease molecules and therapies |
US10738295B2 (en) | 2012-10-04 | 2020-08-11 | Research Development Foundation | Serine protease molecules and therapies |
US10087250B2 (en) | 2012-10-08 | 2018-10-02 | Roche Glycart Ag | Fc-free antibodies comprising two fab-fragments and methods of use |
CN104271602A (zh) * | 2012-11-21 | 2015-01-07 | 武汉友芝友生物制药有限公司 | 双特异性抗体 |
EP2922874A4 (en) * | 2012-11-21 | 2016-10-19 | Wuhan Yzy Biopharma Co Ltd | BISPECIFIC ANTIBODIES |
JP7080955B2 (ja) | 2012-11-28 | 2022-06-06 | ザイムワークス,インコーポレイテッド | 遺伝子操作された免疫グロブリン重鎖-軽鎖対およびその使用 |
KR20150103008A (ko) * | 2012-11-28 | 2015-09-09 | 자임워크스 인코포레이티드 | 가공된 면역글로불린 중쇄-경쇄 쌍 및 이들의 용도 |
KR20220086718A (ko) * | 2012-11-28 | 2022-06-23 | 자임워크스 인코포레이티드 | 가공된 면역글로불린 중쇄-경쇄 쌍 및 이들의 용도 |
US9914785B2 (en) | 2012-11-28 | 2018-03-13 | Zymeworks Inc. | Engineered immunoglobulin heavy chain-light chain pairs and uses thereof |
KR20210070401A (ko) * | 2012-11-28 | 2021-06-14 | 자임워크스 인코포레이티드 | 가공된 면역글로불린 중쇄-경쇄 쌍 및 이들의 용도 |
JP2021048850A (ja) * | 2012-11-28 | 2021-04-01 | ザイムワークス,インコーポレイテッド | 遺伝子操作された免疫グロブリン重鎖−軽鎖対およびその使用 |
EP2925785A4 (en) * | 2012-11-28 | 2016-11-16 | Zymeworks Inc | MANIPULATED IMMUNOGLOBULIN HEAVY CHAIN LIGHT CHAIN COUPLES AND USES THEREOF |
KR102545617B1 (ko) | 2012-11-28 | 2023-06-20 | 자임워크스 비씨 인코포레이티드 | 가공된 면역글로불린 중쇄-경쇄 쌍 및 이들의 용도 |
US11078296B2 (en) | 2012-11-28 | 2021-08-03 | Zymeworks Inc. | Engineered immunoglobulin heavy chain-light chain pairs and uses thereof |
JP2016508117A (ja) * | 2012-11-28 | 2016-03-17 | ザイムワークス,インコーポレイテッド | 遺伝子操作された免疫グロブリン重鎖−軽鎖対およびその使用 |
US10077298B2 (en) | 2012-11-28 | 2018-09-18 | Zymeworks Inc. | Engineered immunoglobulin heavy chain-light chain pairs and uses thereof |
KR102264570B1 (ko) * | 2012-11-28 | 2021-06-14 | 자임워크스 인코포레이티드 | 가공된 면역글로불린 중쇄-경쇄 쌍 및 이들의 용도 |
JP2018162253A (ja) * | 2012-11-28 | 2018-10-18 | ザイムワークス,インコーポレイテッド | 遺伝子操作された免疫グロブリン重鎖−軽鎖対およびその使用 |
KR102411491B1 (ko) | 2012-11-28 | 2022-06-22 | 자임워크스 인코포레이티드 | 가공된 면역글로불린 중쇄-경쇄 쌍 및 이들의 용도 |
CN105026430A (zh) * | 2012-11-28 | 2015-11-04 | 酵活有限公司 | 工程化免疫球蛋白重链-轻链对及其用途 |
US11286293B2 (en) | 2012-11-28 | 2022-03-29 | Zymeworks, Inc. | Engineered immunoglobulin heavy chain-light chain pairs and uses thereof |
US10766960B2 (en) | 2012-12-27 | 2020-09-08 | Chugai Seiyaku Kabushiki Kaisha | Heterodimerized polypeptide |
US10988537B2 (en) | 2013-02-01 | 2021-04-27 | Regeneren Pharmaceuticals, Inc. | Antibodies comprising chimeric constant domains |
US10781257B2 (en) | 2013-02-26 | 2020-09-22 | Roche GlyeArt AG | Bispecific T cell activating antigen binding molecules |
EP2961773B1 (en) * | 2013-02-26 | 2019-03-20 | Roche Glycart AG | Bispecific t cell activating antigen binding molecules |
US10155815B2 (en) | 2013-02-26 | 2018-12-18 | Roche Glycart Ag | Bispecific T cell activating antigen binding molecules |
US11459404B2 (en) | 2013-02-26 | 2022-10-04 | Roche Glycart Ag | Bispecific T cell activating antigen binding molecules |
US10781258B2 (en) | 2013-02-26 | 2020-09-22 | Roche Glycart Ag | Bispecific T cell activating antigen binding molecules |
US10465006B2 (en) * | 2013-07-05 | 2019-11-05 | Genmab A/S | Humanized or chimeric CD3 antibodies |
US20160168247A1 (en) * | 2013-07-05 | 2016-06-16 | Genmab A/S | Humanized or chimeric cd3 antibodies |
WO2015046467A1 (ja) | 2013-09-27 | 2015-04-02 | 中外製薬株式会社 | ポリペプチド異種多量体の製造方法 |
US11124576B2 (en) | 2013-09-27 | 2021-09-21 | Chungai Seiyaku Kabushiki Kaisha | Method for producing polypeptide heteromultimer |
US10323099B2 (en) | 2013-10-11 | 2019-06-18 | Hoffmann-La Roche Inc. | Multispecific domain exchanged common variable light chain antibodies |
US11739149B2 (en) | 2013-11-11 | 2023-08-29 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule containing modified antibody variable region |
CN105940107B (zh) * | 2013-11-11 | 2021-06-15 | 中外制药株式会社 | 含有改变了抗体可变区的抗原结合分子 |
KR20160083094A (ko) * | 2013-11-11 | 2016-07-11 | 추가이 세이야쿠 가부시키가이샤 | 개변된 항체 가변영역을 포함하는 항원 결합 분자 |
CN105940107A (zh) * | 2013-11-11 | 2016-09-14 | 中外制药株式会社 | 含有改变了抗体可变区的抗原结合分子 |
CN113307873A (zh) * | 2013-11-11 | 2021-08-27 | 中外制药株式会社 | 含有改变了抗体可变区的抗原结合分子 |
JP2020196741A (ja) * | 2013-11-11 | 2020-12-10 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子 |
AU2014347565B2 (en) * | 2013-11-11 | 2020-08-13 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule containing modified antibody variable region |
JPWO2015068847A1 (ja) * | 2013-11-11 | 2017-03-09 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子 |
KR102551410B1 (ko) | 2013-11-11 | 2023-07-04 | 추가이 세이야쿠 가부시키가이샤 | 개변된 항체 가변영역을 포함하는 항원 결합 분자 |
WO2015068847A1 (ja) | 2013-11-11 | 2015-05-14 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子 |
US11732054B2 (en) | 2013-12-17 | 2023-08-22 | Genentech, Inc. | Anti-CD3 antibodies and methods of use |
US10640572B2 (en) | 2013-12-17 | 2020-05-05 | Genentech, Inc. | Anti-CD3 antibodies and methods of use |
US11186650B2 (en) | 2013-12-17 | 2021-11-30 | Genentech, Inc. | Anti-CD3 antibodies and methods of use |
US10174124B2 (en) | 2013-12-17 | 2019-01-08 | Genentech, Inc. | Anti-CD3 antibodies and methods of use |
US11530275B2 (en) | 2013-12-17 | 2022-12-20 | Genentech, Inc. | Anti-CD3 antibodies and methods of use |
US10865251B2 (en) | 2013-12-17 | 2020-12-15 | Genentech, Inc. | Anti-CD3 antibodies and methods of use |
US11613575B2 (en) | 2014-01-09 | 2023-03-28 | Genmab A/S | Humanized or chimeric CD3 antibodies |
US10407501B2 (en) * | 2014-01-09 | 2019-09-10 | Genmab A/S | Humanized or chimeric CD3 antibodies |
US11434300B2 (en) | 2014-03-19 | 2022-09-06 | Regeneron Pharmaceuticals, Inc. | Methods and antibody compositions for tumor treatment |
US10550193B2 (en) * | 2014-03-19 | 2020-02-04 | Regeneron Pharmaceuticals, Inc. | Methods and antibody compositions for tumor treatment |
US11485790B2 (en) | 2014-04-07 | 2022-11-01 | Chugai Seiyaku Kabushiki Kaisha | Immunoactivating antigen-binding molecule |
EP3130606B1 (en) | 2014-04-07 | 2021-10-13 | Chugai Seiyaku Kabushiki Kaisha | Immunoactivating bispecific antibodies |
JPWO2015174439A1 (ja) * | 2014-05-13 | 2017-04-20 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子 |
WO2015174439A1 (ja) * | 2014-05-13 | 2015-11-19 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子 |
US11505605B2 (en) | 2014-05-13 | 2022-11-22 | Chugai Seiyaku Kabushiki Kaisha | T cell-redirected antigen-binding molecule for cells having immunosuppression function |
US11306156B2 (en) | 2014-05-28 | 2022-04-19 | Zymeworks Inc. | Modified antigen binding polypeptide constructs and uses thereof |
KR20170044613A (ko) * | 2014-05-28 | 2017-04-25 | 자임워크스 인코포레이티드 | 변형된 항원 결합 폴리펩티드 작제물 및 이의 용도 |
KR102493430B1 (ko) * | 2014-05-28 | 2023-01-31 | 자임워크스 비씨 인코포레이티드 | 변형된 항원 결합 폴리펩티드 작제물 및 이의 용도 |
JP2017524740A (ja) * | 2014-07-26 | 2017-08-31 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | 二重特異性抗体のための精製プラットフォーム |
JP2017522888A (ja) * | 2014-07-29 | 2017-08-17 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | 多重特異性抗体 |
US11117965B2 (en) | 2014-08-04 | 2021-09-14 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
JP2020122013A (ja) * | 2014-08-04 | 2020-08-13 | エンクマフ エスアーエールエル | Cd3イプシロンおよびbcmaに対する二特異性抗体 |
US10611840B2 (en) | 2014-08-04 | 2020-04-07 | Hoffman-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
US10611841B2 (en) | 2014-08-04 | 2020-04-07 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
US9914776B2 (en) | 2014-08-04 | 2018-03-13 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
JP2017532290A (ja) * | 2014-08-04 | 2017-11-02 | エンクマフ アーゲー | Cd3イプシロンおよびbcmaに対する二特異性抗体 |
JP2017525690A (ja) * | 2014-08-04 | 2017-09-07 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | 二重特異性t細胞活性化抗原結合分子 |
US11084877B2 (en) | 2014-09-12 | 2021-08-10 | Genentech, Inc. | Anti-CLL-1 antibodies and immunoconjugates |
KR20170104635A (ko) | 2014-09-26 | 2017-09-15 | 추가이 세이야쿠 가부시키가이샤 | 세포상해 유도 치료제 |
US9975966B2 (en) | 2014-09-26 | 2018-05-22 | Chugai Seiyaku Kabushiki Kaisha | Cytotoxicity-inducing theraputic agent |
JP2016166174A (ja) * | 2014-09-26 | 2016-09-15 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
JP2020141709A (ja) * | 2014-09-26 | 2020-09-10 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
KR20230101934A (ko) | 2014-09-26 | 2023-07-06 | 추가이 세이야쿠 가부시키가이샤 | 세포상해 유도 치료제 |
TWI724889B (zh) * | 2014-09-26 | 2021-04-11 | 日商中外製藥股份有限公司 | 細胞傷害誘導治療劑 |
WO2016047722A1 (ja) * | 2014-09-26 | 2016-03-31 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
JP2016196468A (ja) * | 2014-09-26 | 2016-11-24 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
KR20210121268A (ko) | 2014-09-26 | 2021-10-07 | 추가이 세이야쿠 가부시키가이샤 | 세포상해 유도 치료제 |
JP7054402B2 (ja) | 2014-09-26 | 2022-04-13 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
US11001643B2 (en) | 2014-09-26 | 2021-05-11 | Chugai Seiyaku Kabushiki Kaisha | Cytotoxicity-inducing therapeutic agent |
JP5941230B1 (ja) * | 2014-09-26 | 2016-06-29 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
JP5941235B1 (ja) * | 2014-09-26 | 2016-06-29 | 中外製薬株式会社 | 細胞傷害誘導治療剤 |
US11154615B2 (en) | 2014-11-11 | 2021-10-26 | Chugai Seiyaku Kabushiki Kaisha | Library of antigen-binding molecules including modified antibody variable region |
JPWO2016076345A1 (ja) * | 2014-11-11 | 2017-08-17 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子のライブラリ |
WO2016076345A1 (ja) * | 2014-11-11 | 2016-05-19 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子のライブラリ |
JP2021120375A (ja) * | 2014-11-11 | 2021-08-19 | 中外製薬株式会社 | 改変された抗体可変領域を含む抗原結合分子のライブラリ |
US10662244B2 (en) | 2014-11-17 | 2020-05-26 | Regeneron Pharmaceuticals, Inc. | Methods for tumor treatment using CD3XCD20 bispecific antibody |
US10781262B2 (en) | 2014-11-20 | 2020-09-22 | Hoffmann-La Roche Inc. | Combination therapy of T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists |
US11613587B2 (en) | 2014-11-20 | 2023-03-28 | Hoffmann-La Roche Inc. | Combination therapy of T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists |
US20200199231A1 (en) * | 2015-01-08 | 2020-06-25 | Genmab A/S | Bispecific antibodies against cd3 and cd20 |
WO2016131769A3 (en) * | 2015-02-16 | 2016-12-22 | Lonza Ltd | Cl and/or ch1 mutated antibodies for drug conjugation |
US10953106B2 (en) | 2015-02-16 | 2021-03-23 | Lonza Ltd | Antibodies |
US11833222B2 (en) | 2015-02-16 | 2023-12-05 | Lonza Ltd | CL and/or CH1 mutated antibodies for drug conjugation |
EP3878861A1 (en) * | 2015-02-16 | 2021-09-15 | Lonza Ltd. | Cl and/or ch1 mutated antibodies for drug conjugation |
US11518807B2 (en) | 2015-03-30 | 2022-12-06 | Regeneron Pharmaceuticals, Inc. | Heavy chain constant regions with reduced binding to Fc gamma receptors |
US10556952B2 (en) | 2015-03-30 | 2020-02-11 | Regeneron Pharmaceuticals, Inc. | Heavy chain constant regions with reduced binding to Fc gamma receptors |
US11142587B2 (en) | 2015-04-01 | 2021-10-12 | Chugai Seiyaku Kabushiki Kaisha | Method for producing polypeptide hetero-oligomer |
JP7082484B2 (ja) | 2015-04-01 | 2022-06-08 | 中外製薬株式会社 | ポリペプチド異種多量体の製造方法 |
JPWO2016159213A1 (ja) * | 2015-04-01 | 2018-02-15 | 中外製薬株式会社 | ポリペプチド異種多量体の製造方法 |
US11400157B2 (en) | 2015-05-13 | 2022-08-02 | Chugai Seiyaku Kabushiki Kaisha | Multiple antigen binding molecular fusion, pharmaceutical composition, method for identifying linear epitope, and method for preparing multiple antigen binding molecular fusion |
WO2016182064A1 (ja) * | 2015-05-13 | 2016-11-17 | 中外製薬株式会社 | 多重抗原結合分子融合体、医薬組成物、線状エピトープの同定方法、および多重抗原結合分子融合体の製造方法 |
JPWO2016182064A1 (ja) * | 2015-05-13 | 2018-03-01 | 中外製薬株式会社 | 多重抗原結合分子融合体、医薬組成物、線状エピトープの同定方法、および多重抗原結合分子融合体の製造方法 |
US10501545B2 (en) | 2015-06-16 | 2019-12-10 | Genentech, Inc. | Anti-CLL-1 antibodies and methods of use |
WO2016205200A1 (en) | 2015-06-16 | 2016-12-22 | Genentech, Inc. | Anti-cll-1 antibodies and methods of use |
US10323094B2 (en) | 2015-06-16 | 2019-06-18 | Genentech, Inc. | Humanized and affinity matured antibodies to FcRH5 and methods of use |
US11192950B2 (en) | 2015-06-16 | 2021-12-07 | Genentech, Inc. | Humanized and affinity matured antibodies to FcRH5 and methods of use |
US11466087B2 (en) | 2015-06-16 | 2022-10-11 | Genentech, Inc. | Anti-CLL-1 antibodies and methods of use |
US10766967B2 (en) | 2015-10-02 | 2020-09-08 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
US11161915B2 (en) | 2015-10-08 | 2021-11-02 | Zymeworks Inc. | Antigen-binding polypeptide constructs comprising kappa and lambda light chains and uses thereof |
WO2017086367A1 (ja) | 2015-11-18 | 2017-05-26 | 中外製薬株式会社 | 免疫抑制機能を有する細胞に対するt細胞リダイレクト抗原結合分子を用いた併用療法 |
US11660340B2 (en) | 2015-11-18 | 2023-05-30 | Chugai Seiyaku Kabushiki Kaisha | Combination therapy using T cell redirection antigen binding molecule against cell having immunosuppressing function |
US11649293B2 (en) | 2015-11-18 | 2023-05-16 | Chugai Seiyaku Kabushiki Kaisha | Method for enhancing humoral immune response |
WO2017086419A1 (ja) * | 2015-11-18 | 2017-05-26 | 中外製薬株式会社 | 液性免疫応答の増強方法 |
US11013801B2 (en) | 2015-12-09 | 2021-05-25 | Hoffmann-La Roche Inc. | Treatment method |
US11649262B2 (en) | 2015-12-28 | 2023-05-16 | Chugai Seiyaku Kabushiki Kaisha | Method for promoting efficiency of purification of Fc region-containing polypeptide |
US10596257B2 (en) | 2016-01-08 | 2020-03-24 | Hoffmann-La Roche Inc. | Methods of treating CEA-positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD3 bispecific antibodies |
WO2017159287A1 (ja) | 2016-03-14 | 2017-09-21 | 中外製薬株式会社 | 癌の治療に用いるための細胞傷害誘導治療剤 |
US11072666B2 (en) | 2016-03-14 | 2021-07-27 | Chugai Seiyaku Kabushiki Kaisha | Cell injury inducing therapeutic drug for use in cancer therapy |
US11242390B2 (en) | 2016-03-22 | 2022-02-08 | Hoffmann-La Roche Inc. | Protease-activated T cell bispecific molecules |
JP2019529499A (ja) * | 2016-09-30 | 2019-10-17 | ベイラー カレッジ オブ メディスンBaylor College Of Medicine | 組織指向性発現を用いた抗体ベースの遺伝子治療 |
US10882918B2 (en) | 2016-09-30 | 2021-01-05 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
US11466094B2 (en) | 2016-11-15 | 2022-10-11 | Genentech, Inc. | Dosing for treatment with anti-CD20/anti-CD3 bispecific antibodies |
US11952422B2 (en) | 2017-12-05 | 2024-04-09 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule comprising altered antibody variable region binding CD3 and CD137 |
US11667713B2 (en) | 2017-12-28 | 2023-06-06 | Chugai Seiyaku Kabushiki Kaisha | Cytotoxicity-inducing therapeutic agent |
US11866498B2 (en) | 2018-02-08 | 2024-01-09 | Genentech, Inc. | Bispecific antigen-binding molecules and methods of use |
WO2019160007A1 (ja) | 2018-02-14 | 2019-08-22 | 中外製薬株式会社 | 抗原結合分子および組合せ |
WO2019244973A1 (ja) | 2018-06-20 | 2019-12-26 | 中外製薬株式会社 | 標的細胞に対する免疫反応を活性化する方法およびその組成物 |
US11590223B2 (en) | 2018-08-31 | 2023-02-28 | Regeneron Pharmaceuticals, Inc. | Dosing strategy that mitigates cytokine release syndrome for therapeutic antibodies |
KR20220004087A (ko) | 2019-04-19 | 2022-01-11 | 추가이 세이야쿠 가부시키가이샤 | 항체 개변 부위 인식 키메라 수용체 |
WO2020213724A1 (ja) | 2019-04-19 | 2020-10-22 | 中外製薬株式会社 | 抗体改変部位認識キメラ受容体 |
KR20230011470A (ko) | 2019-06-10 | 2023-01-20 | 추가이 세이야쿠 가부시키가이샤 | 사이토카인 저해제와 조합하여 사용하기 위한 항t세포 항원 결합 분자 |
KR20220019706A (ko) | 2019-06-10 | 2022-02-17 | 추가이 세이야쿠 가부시키가이샤 | 사이토카인 저해제와 조합하여 사용하기 위한 항t세포 항원 결합 분자 |
US11845799B2 (en) | 2019-12-13 | 2023-12-19 | Genentech, Inc. | Anti-Ly6G6D antibodies and methods of use |
US11274151B2 (en) | 2020-03-31 | 2022-03-15 | Chugai Seiyaku Kabushiki Kaisha | CD3-targeting and DLL3-targeting multispecific antigen-binding molecules and uses thereof |
US11718672B2 (en) | 2020-03-31 | 2023-08-08 | Chugai Seiyaki Kabushiki Kaisha | CD137- and DLL3-targeting multispecific antigen-binding molecules |
JP7085666B2 (ja) | 2020-03-31 | 2022-06-16 | 中外製薬株式会社 | Dll3標的多重特異性抗原結合分子およびその使用 |
KR20220161375A (ko) | 2020-03-31 | 2022-12-06 | 추가이 세이야쿠 가부시키가이샤 | 다중 특이성 항원 결합 분자를 제조하기 위한 방법 |
JP2021159081A (ja) * | 2020-03-31 | 2021-10-11 | 中外製薬株式会社 | Dll3標的多重特異性抗原結合分子およびその使用 |
KR20220161156A (ko) | 2020-03-31 | 2022-12-06 | 추가이 세이야쿠 가부시키가이샤 | 면역 활성화 다중 특이성 항원 결합 분자 및 그의 사용 |
US11780920B2 (en) | 2020-06-19 | 2023-10-10 | Hoffmann-La Roche Inc. | Antibodies binding to CD3 and CD19 |
KR20230152789A (ko) | 2020-06-19 | 2023-11-03 | 추가이 세이야쿠 가부시키가이샤 | 혈관신생 저해제와 조합하여 사용하기 위한 항t세포 항원 결합 분자 |
KR20230028225A (ko) | 2020-06-19 | 2023-02-28 | 추가이 세이야쿠 가부시키가이샤 | 혈관신생 저해제와 조합하여 사용하기 위한 항t세포 항원 결합 분자 |
US11845805B2 (en) | 2020-09-10 | 2023-12-19 | Genmab A/S | Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma |
US11858995B2 (en) | 2020-09-10 | 2024-01-02 | Genmab A/S | Bispecific antibodies against CD3 and CD20 for treating chronic lymphocytic leukemia |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6963665B2 (ja) | 細胞傷害誘導治療剤 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11845786 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2012546900 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2819530 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2013/006100 Country of ref document: MX |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20137016886 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2013129694 Country of ref document: RU Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2011337697 Country of ref document: AU Date of ref document: 20111130 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13990088 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112013013311 Country of ref document: BR |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112013013311 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112013013311 Country of ref document: BR Kind code of ref document: A2 Effective date: 20130528 |