WO2022171100A1 - Gpc3人源化抗体及其应用 - Google Patents
Gpc3人源化抗体及其应用 Download PDFInfo
- Publication number
- WO2022171100A1 WO2022171100A1 PCT/CN2022/075588 CN2022075588W WO2022171100A1 WO 2022171100 A1 WO2022171100 A1 WO 2022171100A1 CN 2022075588 W CN2022075588 W CN 2022075588W WO 2022171100 A1 WO2022171100 A1 WO 2022171100A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cancer
- antibody
- antigen
- gpc3
- cells
- Prior art date
Links
- 238000009739 binding Methods 0.000 claims abstract description 134
- 230000027455 binding Effects 0.000 claims abstract description 118
- 108091007433 antigens Proteins 0.000 claims abstract description 112
- 102000036639 antigens Human genes 0.000 claims abstract description 112
- 239000000427 antigen Substances 0.000 claims abstract description 110
- 239000012634 fragment Substances 0.000 claims abstract description 87
- 238000000034 method Methods 0.000 claims abstract description 47
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 46
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 claims abstract description 38
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims abstract description 29
- 231100000844 hepatocellular carcinoma Toxicity 0.000 claims abstract description 28
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 28
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 25
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 23
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 23
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims abstract description 22
- 229940127121 immunoconjugate Drugs 0.000 claims abstract description 21
- 239000013604 expression vector Substances 0.000 claims abstract description 15
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 12
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 102100032530 Glypican-3 Human genes 0.000 claims abstract 17
- 210000004027 cell Anatomy 0.000 claims description 138
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 42
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 36
- 241000282693 Cercopithecidae Species 0.000 claims description 22
- 201000011510 cancer Diseases 0.000 claims description 22
- 230000035772 mutation Effects 0.000 claims description 21
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 21
- 201000010099 disease Diseases 0.000 claims description 20
- 238000006467 substitution reaction Methods 0.000 claims description 18
- 125000000539 amino acid group Chemical group 0.000 claims description 17
- 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 claims description 15
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 15
- 150000001413 amino acids Chemical class 0.000 claims description 13
- 239000012636 effector Substances 0.000 claims description 13
- -1 CD3ζ Proteins 0.000 claims description 10
- 210000000822 natural killer cell Anatomy 0.000 claims description 10
- 239000013598 vector Substances 0.000 claims description 10
- 206010033128 Ovarian cancer Diseases 0.000 claims description 8
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 8
- 238000010494 dissociation reaction Methods 0.000 claims description 8
- 230000005593 dissociations Effects 0.000 claims description 8
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 7
- 238000012217 deletion Methods 0.000 claims description 7
- 230000037430 deletion Effects 0.000 claims description 7
- 206010017758 gastric cancer Diseases 0.000 claims description 7
- 238000003780 insertion Methods 0.000 claims description 7
- 230000037431 insertion Effects 0.000 claims description 7
- 201000011549 stomach cancer Diseases 0.000 claims description 7
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 6
- 206010005949 Bone cancer Diseases 0.000 claims description 6
- 208000018084 Bone neoplasm Diseases 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 6
- 208000026310 Breast neoplasm Diseases 0.000 claims description 6
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 6
- 206010009944 Colon cancer Diseases 0.000 claims description 6
- 206010025323 Lymphomas Diseases 0.000 claims description 6
- 206010029260 Neuroblastoma Diseases 0.000 claims description 6
- 102220545533 Programmed cell death 1 ligand 1_R71A_mutation Human genes 0.000 claims description 6
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 6
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 6
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 6
- 208000008383 Wilms tumor Diseases 0.000 claims description 6
- 201000010881 cervical cancer Diseases 0.000 claims description 6
- 208000029742 colonic neoplasm Diseases 0.000 claims description 6
- 208000006359 hepatoblastoma Diseases 0.000 claims description 6
- 208000032839 leukemia Diseases 0.000 claims description 6
- 206010024627 liposarcoma Diseases 0.000 claims description 6
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 6
- 208000006178 malignant mesothelioma Diseases 0.000 claims description 6
- 201000001441 melanoma Diseases 0.000 claims description 6
- 201000008026 nephroblastoma Diseases 0.000 claims description 6
- 206010038038 rectal cancer Diseases 0.000 claims description 6
- 201000001275 rectum cancer Diseases 0.000 claims description 6
- 102200148949 rs28936686 Human genes 0.000 claims description 6
- 201000000849 skin cancer Diseases 0.000 claims description 6
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 5
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 5
- 206010014733 Endometrial cancer Diseases 0.000 claims description 5
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 5
- 101001079285 Homo sapiens Immunoglobulin heavy joining 1 Proteins 0.000 claims description 5
- 101000998952 Homo sapiens Immunoglobulin heavy variable 1-3 Proteins 0.000 claims description 5
- 101001047625 Homo sapiens Immunoglobulin kappa variable 2-40 Proteins 0.000 claims description 5
- 102100028078 Immunoglobulin heavy joining 1 Human genes 0.000 claims description 5
- 102100036886 Immunoglobulin heavy variable 1-3 Human genes 0.000 claims description 5
- 102100022948 Immunoglobulin kappa variable 2-40 Human genes 0.000 claims description 5
- 102100029193 Low affinity immunoglobulin gamma Fc region receptor III-A Human genes 0.000 claims description 5
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 claims description 5
- 206010061306 Nasopharyngeal cancer Diseases 0.000 claims description 5
- 210000004443 dendritic cell Anatomy 0.000 claims description 5
- 201000010536 head and neck cancer Diseases 0.000 claims description 5
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 5
- 210000002540 macrophage Anatomy 0.000 claims description 5
- 230000001404 mediated effect Effects 0.000 claims description 5
- 210000001616 monocyte Anatomy 0.000 claims description 5
- 210000000581 natural killer T-cell Anatomy 0.000 claims description 5
- 201000003707 ovarian clear cell carcinoma Diseases 0.000 claims description 5
- 229940124597 therapeutic agent Drugs 0.000 claims description 5
- 101100450694 Arabidopsis thaliana HFR1 gene Proteins 0.000 claims description 4
- 206010005003 Bladder cancer Diseases 0.000 claims description 4
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 4
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 4
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 4
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 4
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims description 4
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims description 4
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 4
- 206010060862 Prostate cancer Diseases 0.000 claims description 4
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 4
- 206010038389 Renal cancer Diseases 0.000 claims description 4
- 206010041067 Small cell lung cancer Diseases 0.000 claims description 4
- 208000024313 Testicular Neoplasms Diseases 0.000 claims description 4
- 206010057644 Testis cancer Diseases 0.000 claims description 4
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 4
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 4
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 4
- 230000009977 dual effect Effects 0.000 claims description 4
- 201000004101 esophageal cancer Diseases 0.000 claims description 4
- 201000010982 kidney cancer Diseases 0.000 claims description 4
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 4
- 201000002528 pancreatic cancer Diseases 0.000 claims description 4
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 4
- 208000000587 small cell lung carcinoma Diseases 0.000 claims description 4
- 201000003120 testicular cancer Diseases 0.000 claims description 4
- 201000002510 thyroid cancer Diseases 0.000 claims description 4
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 4
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 3
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 3
- 210000003630 histaminocyte Anatomy 0.000 claims description 3
- 230000004068 intracellular signaling Effects 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000020816 lung neoplasm Diseases 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 210000000440 neutrophil Anatomy 0.000 claims description 3
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 3
- 239000003053 toxin Substances 0.000 claims description 3
- 231100000765 toxin Toxicity 0.000 claims description 3
- 108700012359 toxins Proteins 0.000 claims description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 2
- 102100023990 60S ribosomal protein L17 Human genes 0.000 claims description 2
- 101150075175 Asgr1 gene Proteins 0.000 claims description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims description 2
- 102100027207 CD27 antigen Human genes 0.000 claims description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims description 2
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims description 2
- 101150013553 CD40 gene Proteins 0.000 claims description 2
- 102100024151 Cadherin-16 Human genes 0.000 claims description 2
- 102220600740 Calmodulin-binding transcription activator 1_Q1E_mutation Human genes 0.000 claims description 2
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 claims description 2
- 102100038449 Claudin-6 Human genes 0.000 claims description 2
- 108090000229 Claudin-6 Proteins 0.000 claims description 2
- 108010055196 EphA2 Receptor Proteins 0.000 claims description 2
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 claims description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 claims description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims description 2
- 241000238631 Hexapoda Species 0.000 claims description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 2
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 2
- 101000762246 Homo sapiens Cadherin-16 Proteins 0.000 claims description 2
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 claims description 2
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 claims description 2
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 claims description 2
- 101100334515 Homo sapiens FCGR3A gene Proteins 0.000 claims description 2
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 claims description 2
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 claims description 2
- 101001005719 Homo sapiens Melanoma-associated antigen 3 Proteins 0.000 claims description 2
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims description 2
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 claims description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims description 2
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 claims description 2
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 claims description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 2
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 claims description 2
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 claims description 2
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 claims description 2
- 102100029205 Low affinity immunoglobulin gamma Fc region receptor II-b Human genes 0.000 claims description 2
- 102100025082 Melanoma-associated antigen 3 Human genes 0.000 claims description 2
- 102100034256 Mucin-1 Human genes 0.000 claims description 2
- 102100023123 Mucin-16 Human genes 0.000 claims description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims description 2
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 claims description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 2
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 claims description 2
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims description 2
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 2
- 102100029986 Receptor tyrosine-protein kinase erbB-3 Human genes 0.000 claims description 2
- 101710100969 Receptor tyrosine-protein kinase erbB-3 Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 claims description 2
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 claims description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 2
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 2
- 102000040856 WT1 Human genes 0.000 claims description 2
- 108700020467 WT1 Proteins 0.000 claims description 2
- 101150084041 WT1 gene Proteins 0.000 claims description 2
- 239000012472 biological sample Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 2
- 230000001268 conjugating effect Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 claims description 2
- 210000003515 double negative t cell Anatomy 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 108010087914 epidermal growth factor receptor VIII Proteins 0.000 claims description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 108020005243 folate receptor Proteins 0.000 claims description 2
- 102000006815 folate receptor Human genes 0.000 claims description 2
- 210000002443 helper t lymphocyte Anatomy 0.000 claims description 2
- 150000007857 hydrazones Chemical class 0.000 claims description 2
- 210000004962 mammalian cell Anatomy 0.000 claims description 2
- 238000003032 molecular docking Methods 0.000 claims description 2
- 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 claims description 2
- 210000003289 regulatory T cell Anatomy 0.000 claims description 2
- 102220137027 rs886056062 Human genes 0.000 claims description 2
- 230000009870 specific binding Effects 0.000 claims description 2
- 150000003568 thioethers Chemical class 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims 2
- 201000009030 Carcinoma Diseases 0.000 claims 1
- 238000000375 direct analysis in real time Methods 0.000 claims 1
- 238000012063 dual-affinity re-targeting Methods 0.000 claims 1
- 238000002649 immunization Methods 0.000 claims 1
- 230000003053 immunization Effects 0.000 claims 1
- 239000002955 immunomodulating agent Substances 0.000 claims 1
- 229940121354 immunomodulator Drugs 0.000 claims 1
- 230000002611 ovarian Effects 0.000 claims 1
- 102000010956 Glypican Human genes 0.000 description 93
- 108050001154 Glypican Proteins 0.000 description 93
- 108050007237 Glypican-3 Proteins 0.000 description 92
- 108090000623 proteins and genes Proteins 0.000 description 35
- 102000004169 proteins and genes Human genes 0.000 description 31
- 238000002965 ELISA Methods 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 30
- 238000001514 detection method Methods 0.000 description 22
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 22
- 102000048373 human GPC3 Human genes 0.000 description 21
- 235000001014 amino acid Nutrition 0.000 description 17
- 229920001184 polypeptide Polymers 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- 229940024606 amino acid Drugs 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 241001529936 Murinae Species 0.000 description 11
- 229920002971 Heparan sulfate Polymers 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 229950007906 codrituzumab Drugs 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000283707 Capra Species 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 108010029485 Protein Isoforms Proteins 0.000 description 4
- 102000001708 Protein Isoforms Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 229940072221 immunoglobulins Drugs 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 101100337463 Mus musculus Gpc3 gene Proteins 0.000 description 3
- 241000288906 Primates Species 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 239000012146 running buffer Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 241000251730 Chondrichthyes Species 0.000 description 2
- 108700022150 Designed Ankyrin Repeat Proteins Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 2
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 208000003874 Simpson-Golabi-Behmel syndrome Diseases 0.000 description 2
- 102000013814 Wnt Human genes 0.000 description 2
- 108050003627 Wnt Proteins 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 210000003238 esophagus Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000002381 testicular Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 208000007848 Alcoholism Diseases 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
- 108091023037 Aptamer Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 239000000592 Artificial Cell Substances 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
- 108091016585 CD44 antigen Proteins 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 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
- 108020004414 DNA Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010008177 Fd immunoglobulins Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 102000004961 Furin Human genes 0.000 description 1
- 108090001126 Furin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-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
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-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
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-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
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 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
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010079855 Peptide Aptamers Proteins 0.000 description 1
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 101710204410 Scaffold protein Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000271567 Struthioniformes Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- 102100038126 Tenascin Human genes 0.000 description 1
- 108010008125 Tenascin Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241001416177 Vicugna pacos Species 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 210000002203 alpha-beta t lymphocyte Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 239000003855 balanced salt solution Substances 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 201000005200 bronchus cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 150000002019 disulfides Chemical class 0.000 description 1
- YFQXSOYDIKWVRU-UHFFFAOYSA-N dodecanoic acid;hydrate Chemical compound O.CCCCCCCCCCCC(O)=O YFQXSOYDIKWVRU-UHFFFAOYSA-N 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 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 1
- 238000009472 formulation Methods 0.000 description 1
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229930004094 glycosylphosphatidylinositol Natural products 0.000 description 1
- 101150039713 gpc3 gene Proteins 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 231100000784 hepatotoxin Toxicity 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 108091008104 nucleic acid aptamers Proteins 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 201000003733 ovarian melanoma Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 208000023112 overgrowth syndrome Diseases 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000009520 phase I clinical trial Methods 0.000 description 1
- 238000009521 phase II 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
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
- A61K2239/11—Antigen recognition domain
- A61K2239/13—Antibody-based
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
- A61K2239/21—Transmembrane domain
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
- A61K2239/22—Intracellular domain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—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
- C07K2317/50—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
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
- C07K2317/567—Framework region [FR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to the field of antibodies, in particular, to GPC3 humanized antibodies and applications thereof.
- Glypican-3 is a heparan sulfate (HS) glycoprotein, a member of the heparan sulfate proteoglycan family, which is anchored to the cell membrane by Glypican-3 (GPI) surface.
- the GPC3 core protein includes 580 amino acids and is about 70KD in size. After it is cleaved by Furin, a 40kD amino (N) terminal subunit and a 30kD carboxyl (C) terminal subunit are generated. Disulfide linkages.
- the two HS side chains of GPC3 are bound close to the C-terminus (Takahiro Nishida, Hiroaki Kataoka. Glypican3-Targeted Therapy in Hepatocellular Carcinoma, Cancers 2019; 11(9):1339).
- GPC3 plays an important regulatory role in cell proliferation in embryonic mesoderm tissue, and deletion of the GPC3 gene results in an overgrowth syndrome, Simpson-Golabi-Behmel syndrome (SGBS).
- GPC3 was significantly expressed throughout the fetal period, but was not significantly expressed in other normal tissues except for the weak expression in placenta, mammary gland, mesothelial, ovary, lung and kidney tissues after birth to adulthood.
- GPC3 is abnormally expressed in various adult tumor tissues, such as hepatocellular carcinoma (HCC), lung squamous cell carcinoma, gastric cancer, ovarian cancer, etc.
- HCC hepatocellular carcinoma
- lung squamous cell carcinoma gastric cancer
- ovarian cancer etc.
- Codrituzumab (also known as GC33 antibody) is a recombinant humanized monoclonal antibody developed by Chugai Pharmaceuticals in Japan. It binds to the membrane-proximal region of GPC3 protein.
- the GC33 antibody targets GPC3-positive HCC cells and can produce antibody-dependent cytotoxicity (ADCC).
- ADCC antibody-dependent cytotoxicity
- Codrituzumab showed good immune tolerance and had antitumor effects in HCC patients (Ikeda M, Ohkawa S, Okusaka T, et al. Japanese phase I study of GC33, a humanized antibody against glypican- 3for advanced hepatocellular carcinoma. Cancer Sci. 2014, 105, 455–462).
- codrituzumab was less effective than a control group, and researchers believe that patient outcomes could be improved in two ways: using high-dose codrituzumab or Select patients expressing higher levels of GPC3 or CD16 (Abou-Alfa G.K, Puig O, Daniele B, et al. Randomized phase II placebo controlled study of codrituzumab in previously treated patients with advanced hepatocellular carcinoma. J. Hepatol. 2016, 65, 289–295 ). In conclusion, the clinical application of this antibody remains to be discussed.
- the purpose of the present invention is to provide a humanized antibody or antigen-binding fragment that specifically binds to GPC3 in view of the problems existing in the prior art.
- the present invention also provides immunoconjugates, chimeric antigen receptors, immunocompetent cells, multispecific molecules, nucleic acid molecules, expression vectors, host cells, pharmaceutical compositions, methods of preparation, and use.
- an antibody or antigen-binding fragment that specifically binds to GPC3, comprising a humanized heavy chain variable region and/or a humanized light chain variable region, the heavy chain
- the variable region comprises a heavy chain framework region of human origin, a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:15, a CDR2 comprising the amino acid sequence of SEQ ID NO:16, and a CDR2 comprising the amino acid sequence of SEQ ID NO:17 CDR3 of the amino acid sequence
- the light chain variable region comprises a light chain framework region of human origin, a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 18, 21, and the amino acid sequence comprising SEQ ID NO: 19 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20.
- the heavy chain framework region comprises the framework regions HFR1, HFR2 and HFR3 of IGHV1-3*01 set forth in SEQ ID NO:11, and the framework region HFR4 of IGHJ1*01 set forth in SEQ ID NO:12;
- the light chain framework region comprises the framework regions LFR1, LFR2 and LFR3 of IGKV2-40*01 shown in SEQ ID NO: 9, and the framework region LFR4 of IGKJ2*01 shown in SEQ ID NO: 10, wherein the framework region Determined according to the Kabat numbering system.
- the heavy chain framework region comprises a mutation in amino acid residues selected from the group consisting of positions 1, 44, 69, 71, 73, 93, numbered according to the Kabat numbering system, preferably, the mutation comprises: Q1E , R71A and A93T; Q1E, I69L, R71A and A93T; or Q1E, R44G, I69L, R71A, T73K and A93T.
- the light chain framework region comprises at most one amino acid residue mutation, numbered according to the Kabat numbering system; preferably, the mutation is a mutation of an amino acid residue selected from the group consisting of amino acid residues at position 2; preferably, the Mutated to I2V.
- the antibody or antigen-binding fragment comprises: (1) the heavy chain variable region set forth in any one of SEQ ID NOs: 4-6, or, having the same variable region as any one of SEQ ID NOs: 4-6 Sequences with at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity compared to the sequences shown in the item; or, having at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 occurrences compared to the sequence shown in any one of SEQ ID NOs: 4-6 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutated sequences; the mutations may be selected from insertions, deletions and/or substitutions, the Substitutions are preferably conservative amino acid substitutions;
- the light chain variable region shown in any one of SEQ ID NOs: 7-8, 13-14 or, having the light chain variable region shown in any one of SEQ ID NOs: 7-8, 13-14 sequences that are at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequences; or, have Occurs up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 compared to the sequences set forth in any of SEQ ID NOs: 7-8, 13-14 sequences of 1, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations; the mutations may be selected from insertions, deletions and/or substitutions, whereby The substitutions are preferably conservative amino acid substitutions.
- the antibody or antigen-binding fragment comprises: (1) a heavy chain variable region having the sequence shown in SEQ ID NO: 4, 5 and 6, and a light chain variable region having the sequence shown in SEQ ID NO: 14 district; or,
- the antibody or antigen-binding fragment comprises at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95% compared to the CDR1, CDR2 and/or CDR3 , 96%, 97%, 98%, 99% or 100% identical sequences; or 1, 2, 3 or more amino acid insertions, deletions and/or compared to said CDR1, CDR2 and/or CDR3 Sequences of substitutions, preferably conservative amino acid substitutions.
- the antibody or antigen-binding fragment specifically binds to human and/or monkey GPC3 protein; preferably, the dissociation constant (KD) with human and/or monkey GPC3 is not greater than 1.00E-7M, 1.00E -8M, 2.00E-8M, 3.00E-8M, 4.00E-8M, 5.00E-8M, 6.00E-8M, 7.00E-8M, 8.00E-8M, 9.00E-8M, 1.00E-9M, 2.00E -9M, 3.00E-9M, 4.00E-9M, 5.00E-9M, 6.00E-9M, 7.00E-9M, 8.00E-9M, 9.00E-9M or 6.00E-10M.
- KD dissociation constant
- the antibody or antigen-binding fragment is selected from the group consisting of full-length antibodies, VH single-domain structure antibodies, Fab fragments, Fab' fragments, F(ab)'2 fragments, Fd fragments, Fv fragments, complementarity determining regions ( CDR) fragment, single chain variable fragment (scFv), scFV2, disulfide stabilized variable fragment (dsFv), domain antibody, bivalent single chain antibody, single chain phage antibody, bispecific diabody, triple chain Antibody, tetrabody or antibody minimum recognition unit.
- the present invention provides an immunoconjugate comprising any one of the above-mentioned antibodies or antigen-binding fragments and an effector molecule; preferably, the effector molecule is linked to the antibody or antigen-binding fragment.
- the effector molecule comprises a therapeutic agent or a label; preferably, the therapeutic agent is selected from a drug, a toxin, a radioisotope, a chemotherapeutic agent or an immunomodulatory agent.
- the immunoconjugate further comprises a linker for conjugating the effector molecule to the antibody or antigen-binding fragment, the linker including, but not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
- the invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain comprising The antibody or antigen-binding fragment of any one of the above.
- CAR chimeric antigen receptor
- the present invention provides an immunocompetent cell expressing any of the above-mentioned chimeric antigen receptors or comprising a nucleic acid molecule encoding any of the above-mentioned chimeric antigen receptors; preferably, the immunocompetent cells Selected from: T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, the T cells The cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells.
- the present invention provides a multispecific molecule comprising any of the above-mentioned antibodies or antigen-binding fragments; preferably, the multispecific molecule further comprises an antigen that specifically binds to GPC3 or binds to an antigen other than the above-mentioned Any antibody or antigen-binding fragment of a different GPC3 epitope.
- the antigen other than GPC3 is an antigen on the surface of T cells, B cells, natural killer cells, dendritic cells, macrophages, monocytes or neutrophils; preferably, the Antigens other than GPC3 are selected from: CD3, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD16, CD16A, CD32B, PD-1, PD-2, PD-L1, VEGF, NKG2D, CD19, CD20, CD40, CD47, 4-1BB , CD137, EGFR, EGFRvIII, TNF-alpha, CD33, HER2, HER3, HAS, CD5, CD27, EphA2, EpCAM, MUC1, MUC16, CEA, Claudin18.2, folate receptor, Claudin6, WT1, NY-ESO-1 , MAGE3, ASGPR1 or CDH16.
- the multispecific molecule is a tandem scFv, diabody (Db), single chain diabody (scDb), dual affinity retargeting (DART) antibody, F(ab')2, dual Variable domain (DVD) antibodies, Knock in the hole (KiH) antibodies, Docking and Locking (DNL) antibodies, chemically cross-linked antibodies, heteromultimeric antibodies or heteroconjugate antibodies.
- the present invention provides an isolated nucleic acid molecule encoding any of the above-mentioned antibodies or antigen-binding fragments, any of the above-mentioned chimeric antigen receptors, or any of the above-mentioned multispecific molecules.
- the present invention provides a vector comprising the above-described nucleic acid molecule.
- the present invention provides a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, preferably, the host cell is a prokaryotic cell or a eukaryotic cell, including bacteria (Escherichia coli), fungi (yeast) , insect cells or mammalian cells (CHO cell line or 293 cell line).
- bacteria Esscherichia coli
- fungi fungi
- insect cells or mammalian cells
- mammalian cells CHO cell line or 293 cell line.
- the present invention provides a method for preparing any of the above-mentioned antibodies or antigen-binding fragments or multispecific molecules, the method comprising: culturing the above-mentioned host cells, and isolating the antibodies or antigen-binding fragments expressed by the cells, or isolating multispecific molecules expressed by the cells.
- the present invention provides a method for preparing the immunocompetent cells, comprising: introducing into the immunocompetent cells a nucleic acid fragment comprising any one of the above-mentioned chimeric antigen receptors, optionally, the method It also includes enabling the immunocompetent cells to express any one of the above-mentioned chimeric antigen receptors.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a therapeutically effective amount of one or a combination of: an antibody or antigen-binding fragment of any of the foregoing; or an immunoconjugate of any of the foregoing; or any of the foregoing an immunocompetent cell; or any of the above-mentioned multispecific molecules; or any of the above-mentioned nucleic acid molecules, expression vectors or host cells, or a product prepared by the method described in any of the above-mentioned methods, and pharmaceutically acceptable accepted vector.
- the present invention also provides any of the above-mentioned antibodies or antigen-binding fragments, immunoconjugates, immunocompetent cells, multispecific molecules, nucleic acid molecules, expression vectors, products prepared by the method, or Use of a pharmaceutical composition in the preparation of a medicament for treating GPC3-mediated tumors; preferably, the tumor is selected from hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, hepatoblastoma, neuroblastoma, and Wilms Tumor, small cell lung cancer, lung adenocarcinoma, stomach cancer, colon cancer, rectal cancer, cervical cancer, breast cancer, ovarian cancer, skin cancer, lymphoma, prostate cancer, pancreatic cancer, kidney cancer, esophageal cancer, thyroid cancer, testicular cancer Cancer, bladder cancer, bronchial cancer, nasopharyngeal cancer, head and neck cancer, endometrial cancer, brain cancer, bone cancer, leukemia, malignant mesothelio
- the present invention also provides a method of treating a subject having a GPC3-mediated tumor, comprising selecting a subject having a GPC3-expressing cancer, and administering to the subject a therapeutically effective amount Any of the above-mentioned antibodies or antigen-binding fragments, immunoconjugates, immunocompetent cells, multispecific molecules, nucleic acid molecules, expression vectors, products or pharmaceutical compositions prepared by the method; preferably, the tumor selected From hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, hepatoblastoma, neuroblastoma, Wilms tumor, small cell lung cancer, lung adenocarcinoma, gastric cancer, colon cancer, rectal cancer, cervical cancer, breast cancer , ovarian cancer, skin cancer, lymphoma, prostate, pancreas, kidney, esophagus, thyroid, testicular, bladder, bronchus, nasopharyngeal, head and neck, endometrial, brain, bone Cancer
- the present invention also provides any of the above-mentioned antibodies or antigen-binding fragments, immunoconjugates, immunocompetent cells, multispecific molecules, nucleic acid molecules, expression vectors, products or pharmaceutical combinations prepared by the method
- a substance for the treatment of GPC3-positive tumors or cancers preferably, the tumor is selected from hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, hepatoblastoma, neuroblastoma, Wilms tumor, small cell lung cancer, Lung adenocarcinoma, stomach cancer, colon cancer, rectal cancer, cervical cancer, breast cancer, ovarian cancer, skin cancer, lymphoma, prostate, pancreas, kidney, esophagus, thyroid, testicular, bladder, bronchus Cancer, nasopharyngeal cancer, head and neck cancer, endometrial cancer, brain cancer, bone cancer, leukemia, malignant mesothelioma, liposarcoma and other diseases;
- the present invention also provides a kit comprising any of the above-mentioned antibodies or antigen-binding fragments, any of the above-mentioned immunoconjugates, the above-mentioned immunocompetent cells, and any of the above-mentioned multispecific molecules, The above-mentioned nucleic acid molecule, the above-mentioned expression vector, the product prepared according to any one of the above-mentioned methods, or the above-mentioned pharmaceutical composition.
- the present invention also provides the use of any of the above-mentioned antibodies or antigen-binding fragments in the preparation of a reagent for detecting or diagnosing tumors with high GPC3 expression.
- the present invention also provides a method for detecting GPC3 expression in a biological sample, characterized in that the sample from a subject is brought into contact with any of the above-mentioned antibodies or antigen-binding fragments, and the antibody is detected or binding of antigen-binding fragments to the sample.
- GPC3 members of the Glypican family of heparan sulfate (HS) proteoglycans, which are mediated by glycosylphosphatidyl Inositol anchors are attached to the cell surface.
- HS heparan sulfate
- Human GPC3 has four known isoforms (isoforms 1-4) whose nucleic acid and amino acid sequences are known, including GenBank accession numbers: NM_001164617 and NP_001158089 (isoform 1); NM_004484 and NP_004475 (subtype 2); NM_001164618 and NP_001158090 (subtype 3); and NM_001164619 and NP_001158091 (subtype 4).
- the antibodies disclosed herein bind one or more of the four GPC3 isoforms, or conservative variants thereof.
- the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
- antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
- Antibody mimetic refers to an organic compound or binding domain that can specifically bind to an antigen, but is unrelated to the structure of an antibody.
- antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamer or Kunitz-type domain peptide.
- antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to enable specific binding to an antigen or peptide combination.
- Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
- Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, eg, biocompatible polymers.
- Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
- antibody herein includes a typical "quad-chain antibody”, which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); heavy chain refers to a polypeptide chain that is In the N-terminal to C-terminal direction consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and , when the antibody is of the IgE isotype, optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant region in the N-terminal to C-terminal direction Polypeptide chain composed of (CL); heavy chain and heavy chain, heavy chain and light chain are connected by disulfide bonds to form a "Y"-shaped structure.
- immunoglobulins Due to the different amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain, its antigenicity is also different. Accordingly, the "immunoglobulins" herein can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ and ⁇ chains, respectively. , ⁇ chain, ⁇ chain and ⁇ chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
- IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2.
- Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
- Each of the five classes of Ig can have a kappa chain or a lambda chain.
- antibody herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents and vertebrates, eg camelid, ram Camelids, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
- antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies, multivalent antibodies, whole antibodies, antigen-binding fragments, naked antibodies , conjugated antibodies, humanized antibodies or fully human antibodies.
- antigen-binding fragment refers to one or more antibody fragments that retain the ability to specifically bind a target antigen.
- the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
- Antibody fragments can be Fab, F(ab')2, scFv, SMIP, diabodies, tribodies, affibodies, Nanobodies, aptamers or domain antibodies.
- binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment comprising two Fab fragments connected at the hinge region by disulfide bonds; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains consisting of an antibody one-arm Constituent Fv fragments; (v) dAbs comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989) or VHH; (vii) consisting of VH or dAb composed of VL domains; (viii) isolated complementarity determining regions (CDRs); (ix) heavy chain antibody fragments composed of VHH and CH2, CH3; and (x) two or more
- the two domains of the Fv fragment, VL and VH are encoded by separate genes, the two domains can be joined using recombinant methods by a linker that enables it to be made in which the VL and VH regions are paired to form A single protein chain of a monovalent molecule (called a single-chain Fv (scFv); see, eg, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 , 1988).
- scFv single-chain Fv
- These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
- Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
- the term "monoclonal antibody” herein refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and is not limited to the method by which the antibody is produced.
- multispecific herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen combine.
- terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
- valency herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule.
- the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule the existence of points.
- scFv single-chain variable fragment
- linker see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
- Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
- Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
- GGGGS linker with the amino acid sequence
- Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res.
- a disulfide bond may also exist between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).
- humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
- CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
- Humanized antibodies typically retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune responses, and the like.
- variable region herein refers to the region of an antibody heavy or light chain that is involved in binding an antibody to an antigen.
- "Heavy chain variable region” is used interchangeably with “VH” and “HCVR”.
- VL is used interchangeably with "VL”, “LCVR”.
- the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
- a single VH or VL domain may be sufficient to confer antigen binding specificity.
- complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the variable region of the heavy chain (VH) or the hypervariable region (HVR) of the light chain variable region (VL), which is spatially structured It can form precise complementarity with the antigenic epitope, so it is also called the complementarity determining region.
- VH variable region of the heavy chain
- HVR hypervariable region of the light chain variable region
- HCDR variable region of the heavy chain
- LCDR light chain variable region
- frame region or "FR region” are used interchangeably and refer to those amino acid residues other than the CDRs in the variable region of the heavy or light chain of an antibody
- HFR may refer to the framework of the variable region of the heavy chain region
- LFR may refer to the framework region of the light chain variable region.
- CDRs may be labeled and defined by means known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including, but not limited to, the AbRSA website (http://cao.labshare.
- CDRs herein include overlaps and subsets of amino acid residues differently defined.
- the numbering of amino acid residues in "antibodies” or “antigen-binding fragments” described herein is determined by the Kabat numbering system, as detailed in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991, for the numbering of amino acid residues in the variable region of antibodies, please refer to the following website: http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi.
- LFR1 is DIVMTQTPLSLPVTPGEPASISC (SEQ ID NO: 22)
- LFR2 is WYLQKPGQSPQLLIY (SEQ ID NO: 23)
- LFR3 is GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 24)
- LFR4 is FGQGTKLEIK (SEQ ID NO: 10)
- HFR1 is QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 25)
- HFR2 is WVRQAPGQRLEWMG (SEQ ID NO: 26)
- HFR3 is RVTITRDTSASSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 27)
- HFR4 is WGQGTLVTVSS (SEQ ID NO: 12)
- amino acids generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
- amino acids within each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group belong to conservative amino acid substitutions:
- identity and “sequence identity” are used interchangeably herein and are calculated by: To determine the percent "identity” of two amino acid sequences or two nucleic acid sequences, the sequences are optimally Alignment for comparison purposes (eg, gaps may be introduced in either or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
- immunoconjugate refers to a polypeptide molecule comprising at least one effector molecule and at least one antibody or functional fragment thereof.
- effector molecule herein is a portion of an immunoconjugate that is intended to have a desired effect on the cells targeted by the immunoconjugate. Effector molecules are also referred to as effector moieties (EMs), therapeutic or diagnostic agents or tracers or similar terms.
- EMs effector moieties
- chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immunocompetent cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen binding domain, eg, an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immunocompetent cells, and (3) the intracellular signaling domain.
- CARs can utilize extracellular antigen-binding domains to redirect T cells and other immunocompetent cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
- multispecific molecule herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or a different epitope of a different antigen. Bit binding.
- references such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
- immunocompetent cells refers to cells that are responsible for immune functions in an organism.
- immunocompetent cells include lymphocyte-like cells such as T cells, natural killer cells (NK cells), and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; neutrophils, Granulocytes such as eosinophils, basophils, mast cells, etc.
- T cells or NK cells derived from mammals such as humans, dogs, cats, pigs, and mice, and preferably T cells or NK cells derived from humans.
- T cells can be isolated and purified from body fluids such as blood and bone marrow fluid, tissues such as spleen, thymus, lymph nodes, or immunocompetent cells that have infiltrated into cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites.
- T cells produced from ES cells and iPS cells are used. Examples of the T cells include ⁇ - ⁇ T cells, ⁇ - ⁇ T cells, CD8+ T cells, CD4+ T cells, tumor-infiltrating T cells, memory T cells, naive T cells, and NKT cells. It should be noted that the source of the immunocompetent cells and the administration target may be the same or different.
- the administration subject is a human
- immunocompetent cells autologous cells collected from the patient to be administered may be used, or allogeneic cells obtained from other people may be used. That is, the donor and the acceptor may or may not be identical, but are preferably identical.
- Vector refers to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell.
- a vector may contain nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication.
- the vector may also contain one or more selectable marker genes and other genetic elements known in the art.
- host cell herein refers to a cell, which may be a prokaryotic cell or a eukaryotic cell, in which a vector can proliferate and its DNA can be expressed.
- the term also includes any progeny of the subject host cell. It should be understood that not all progeny are identical to the parental cell, and such progeny are included due to the possibility of mutation during replication.
- pharmaceutically acceptable carrier herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible.
- the nature of the carrier will depend upon the particular mode of administration employed.
- parenteral formulations typically contain injectable fluids comprising pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like as a vehicle.
- conventional nontoxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
- compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents, and the like, such as sodium acetate or sorbitan monohydrate Laurate.
- non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents, and the like, such as sodium acetate or sorbitan monohydrate Laurate.
- a therapeutically effective amount refers to an amount of an anti-GPC3 antibody or composition as disclosed herein that is effective to "treat” a disease or disorder in an individual.
- a therapeutically effective amount of an anti-GPC3 antibody or composition as disclosed herein can reduce the number of cancer cells; reduce tumor size or weight; inhibit (ie, slow to some extent and preferably prevent) cancer cell infiltration To peripheral organs; inhibit (ie, to some extent slow and preferably prevent) tumor metastasis; to some extent inhibit tumor growth; and/or to some extent alleviate one or more symptoms associated with cancer.
- an anti-GPC3 antibody or composition as disclosed herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.
- the therapeutically effective amount is a growth inhibitory amount.
- a therapeutically effective amount is an amount that prolongs survival of the patient.
- a therapeutically effective amount is an amount that improves progression-free survival in a patient.
- treatment refers to a method for obtaining beneficial or desired results, including clinical results.
- beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction in the extent of the disease, stabilization of the disease (eg, prevention or delay of the disease) exacerbation), preventing or delaying the spread of the disease (eg, metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the state of the disease, bringing about a remission (partial or complete) of the disease, reducing the need for one or more other drugs to treat the disease dose, delay disease progression, increase or improve quality of life, increase weight gain and/or prolong survival.
- Treatment also encompasses a reduction in the pathological outcome of the cancer (eg, tumor volume). The methods of the present invention encompass any one or more of these therapeutic aspects.
- subject refers to an organism receiving treatment for a particular disease or disorder as described herein.
- subjects and patients include mammals, such as humans, primates (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
- diagnostic herein refers to identifying the presence or nature of a pathological condition such as, but not limited to, liver cancer, ovarian cancer, melanoma, or lung cancer. Diagnostic methods vary in sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of affected individuals that test positive (the percentage of true positives). The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those individuals who test positive without the disease. Although a particular diagnostic method may not provide a definitive diagnosis of the disorder, it is sufficient that the method provides a positive indication to aid in the diagnosis.
- hepatocellular carcinoma herein refers to a primary hepatic malignancy that typically occurs in patients with an inflamed liver caused by viral hepatitis, hepatotoxin, or cirrhosis (often caused by alcoholism). middle. HCC is also known as malignant hepatoma.
- Fig. 1A shows the binding reaction of ELISA detection control antibody and human GPC3-His protein
- Fig. 1B shows the binding reaction of ELISA detection control antibody and monkey GPC3-His protein
- Figure 1C shows the binding reaction of ELISA detection control antibody and mouse GPC3-His protein
- Fig. 2 is the binding reaction of ELISA detection control antibody and polypeptide GC3pep protein
- Figure 3 shows the FACS results of Y035 antibody and T2-23 antibody detecting the expression of GPC3 in HepG2 cells
- Figure 4 is the FACS result of Y035 antibody detecting GPC3 expression in CHO-K1-human GPC3 cells
- Figure 5 is the FACS result of Y035 antibody detecting GPC3 expression in HEK293T-monkey GPC3 cells
- Fig. 6 is ELISA to detect the binding reaction of anti-GPC3 humanized antibody and human GPC3-his protein
- Figure 7A is FACS detection of the binding reaction of anti-GPC3 humanized antibody to CHO-K1-human GPC3 cells
- Fig. 7B is FACS detection of the binding reaction of anti-GPC3 humanized antibody to CHO-K1 cells
- Figure 8A is FACS detection of the binding reaction of anti-GPC3 humanized antibody to HepG2 tumor cells
- FIG. 8B shows the binding reaction of anti-GPC3 humanized antibody to A431 tumor cells detected by FACS
- Fig. 9 is ELISA to detect the binding reaction of anti-GPC3 humanized antibody and murine GPC3-his protein
- Fig. 10 is that ELISA detects the binding reaction of anti-GPC3 humanized antibody and monkey GPC3-His protein
- Figure 11A is FACS detection of the binding reaction of anti-GPC3 humanized antibody to HEK293T-monkey GPC3 cells
- FIG. 11B is FACS detection of the binding reaction of anti-GPC3 humanized antibody to HEK293T cells
- Figure 12 shows the binding reaction of anti-GPC3 humanized antibody and GC3pep polypeptide protein detected by ELISA.
- Example 1 Preparation of control antibodies, preparation of human polypeptides, identification of endogenous cells and preparation of overexpressing cell lines
- the Y035 and T2-23 clones are antibodies that recognize human GPC3 protein, and both have strong binding affinity to human GPC3 protein, and can also bind to GPC3 high-expressing cell lines, such as HepG2.
- the heavy chain variable region and light chain variable region sequences of the Y035 and T2-23 clones were obtained according to patent US2019/0046659A1.
- VL and VH of clones Y035 and T2-23, which recognize human GPC3, and human IgG1 Fc are connected in the order from N-terminus to C-terminus, wherein VH and VL are connected by 3 GGGGS linkers to form scFv-human IgG1 Fc (scFv-hFc), the corresponding nucleotide sequences were cloned into pTT5 vector (purchased from Youbao Bio), and plasmids were prepared according to established standard molecular biology methods. For specific methods, see Sambrook, J. , Fritsch, EF, and Maniatis, T. (1989).
- the expression vector was transiently transfected into HEK293E cells (purchased from the Cell Bank of the Type Culture Collection, Chinese Academy of Sciences) according to the instructions of PEI (purchased from Polysciences) and cultured at 37°C for 5 days using FreeStyle TM 293 (Invitrogen), and the cell components were removed by centrifugation. , the culture supernatant containing the antibody in the form of ScFv-human IgG1 Fc (hFc) was obtained.
- the culture supernatant was loaded onto a protein A chromatography column (Protein A packing AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borgron), washed with PBS phosphate buffer (pH 7.4), and then Wash with 20 mM PB, 1 M NaCl, pH 7.2, and finally eluate with citrate buffer pH 3.4, collect the Fc-tagged antibody eluted from the Protein A column, and use 1/10 volume of pH 8 .0 1M Tris was neutralized, dialyzed overnight at 4°C with PBS, the concentration of the dialyzed antibody was determined by Nanodrop, the purity of the antibody was determined by HPLC-SEC, and the endotoxin detection kit (purchased from Andus) was used to detect the content of the antibody.
- Table 1 is the sequence information of the control antibody, in which the Y035 scFv-hFc position 1-113 is the VL sequence, the 129-243 position is the VH sequence, the T2-23 scFv-hFc position 1-111 is the VL sequence, and the 127- Bit 247 is the VH sequence, underlined.
- the control antibody was compared with human GPC3-His protein (purchased from Acro, product number: GP3-H52H4), monkey GPC3-His protein (purchased from Acro, product number: GP3-C5225) and murine GPC3-His protein (purchased from Sino Biological, product number: 50989-M08B) binding activity was detected by ELISA.
- the specific method is: dilute the antigen protein with PBS to a final concentration of 1 ⁇ g/mL, and then add 50 ⁇ l per well to a 96-well ELISA plate.
- TMB substrate 50 ⁇ l was added to each well, and after 10 minutes of incubation at room temperature, 50 ⁇ l of stop solution (1.0 M HCl) was added to each well.
- OD450nm values were read with an ELISA plate reader (Multimode Plate Reader, EnSight, available from Perkin Elmer).
- the results are shown in Tables 2, 3, 4 and Figures 1A, 1B and 1C, Y035, T2-23 antibodies have good binding activity to human GPC3 protein and monkey GPC3 protein , Y035 does not bind to mouse GPC3 protein, T2-23 Binds well to murine GPC3 protein .
- the IgG isotype control was human IgG1.
- the polypeptide GC3pep (AELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLK, SEQ ID NO:3) of human GPC3 (NCBI:NM_004484.3, Ala524-Lys563) was produced.
- the prepared above-mentioned polypeptides were respectively detected with positive control antibodies that recognized different epitopes according to the ELISA method of Example 1 (A).
- GC3pep indicating that the antigenic polypeptide with binding activity to the antibody has been prepared .
- HepG2 cells were expanded and cultured in a T-75 cell culture flask to the logarithmic growth phase, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS.
- Y035 and T2-23 antibodies were used as primary antibodies, and FITC-labeled secondary antibodies (purchased from Invitrogen, product number: A11013) were detected and analyzed by FACS (FACS CantoTM, purchased from BD Company). The results are shown in Table 6 and Figure 3, indicating that HepG2 cells can bind to both Y035 and T2-23.
- the nucleotide sequence encoding the full-length amino acid sequence of human GPC3 was cloned into pcDNA3.1 vector (purchased from Clontech) and a plasmid was prepared.
- the CHO-K1 cell line purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences
- plasmids 3000 Transfection Kit, purchased from Invitrogen, Cat. No. L3000-015
- DMEM/F12 medium containing 10% (w/w) fetal bovine serum containing 10 ⁇ g/mL puromycin
- Y035 antibody and goat anti-human IgG H+L antibody goat anti-human IgG H+L antibody
- Table 7 illustrates that a series of human GPC3-positive CHO-K1 monoclonal cell lines have been prepared.
- the abscissa is the cell fluorescence intensity, and the ordinate is the number of cells.
- the human GPC3 high-level expressing cell line used in the present invention is 2B5.
- the nucleotide sequence encoding the full-length amino acid sequence of monkey GPC3 (NCBI: XP_011739317.1) was cloned into pcDNA3.1 vector (purchased from Thermofisher scientific) and a plasmid was prepared.
- pcDNA3.1 vector purchased from Thermofisher scientific
- HEK293T cell line Promega, Cat. No.: #E2311
- DMEM medium containing 10 ⁇ g/mL puromycin and 10% (w/w) fetal bovine serum after transfection with Y035 antibody and goat antibody.
- Human IgG H+L antibody (Jackson, Cat.
- the heavy chain and light chain variable region germline genes with high homology to murine antibodies were selected respectively.
- the CDRs of the murine antibodies were grafted into the corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
- the key amino acids in the backbone sequence are back-mutated to the amino acids corresponding to the mouse-derived antibody to ensure the original affinity, that is, a humanized anti-GPC3 monoclonal antibody is obtained.
- the CDR amino acid residues of antibodies are generally identified and annotated by the Kabat numbering system.
- the humanized light chain templates are IGKV2-40*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ1*01.
- the CDRs of the mouse GPC3 antibody were transplanted into the human template, namely Obtain the corresponding humanized version.
- the key amino acids in the FR region sequence of the anti-GPC3 humanized antibody were back-mutated to the amino acids corresponding to the murine antibody to ensure the original affinity.
- the specific back-mutation design is shown in Table 8.
- Grafted represents the insertion of murine antibody CDRs into the human germline FR region sequence; I2V represents the mutation of Grafted position 2 I back to V, and so on.
- the numbering of backmutated amino acids is determined by the Kabat numbering system.
- variable region of the humanized antibody against GPC3 is as follows:
- amino acid sequence of GPC3.VH1 is shown in SEQ ID NO: 4:
- amino acid sequence of GPC3.VH2 is shown in SEQ ID NO: 5:
- amino acid sequence of GPC3.VH5 is shown in SEQ ID NO: 6:
- amino acid sequence of GPC3.VL1 is shown in SEQ ID NO: 7:
- amino acid sequence of GPC3.VL2 is shown in SEQ ID NO: 8:
- amino acid sequence of humanized light chain template IGKV2-40*01 is shown in SEQ ID NO: 9:
- amino acid sequence of humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 10:
- amino acid sequence of humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 11:
- amino acid sequence of humanized heavy chain template IGHJ1*01 is shown in SEQ ID NO: 12:
- the anti-GPC3 antibody has a site NG that is prone to chemical modification, and we point mutated the NG to eliminate the risk of modification.
- amino acid sequence of GPC3.VL1a is shown in SEQ ID NO: 13:
- amino acid sequence of GPC3.VL2a is shown in SEQ ID NO: 14:
- the present invention selects different light chain and heavy chain sequences for cross-combination from the back mutation design of the light chain and heavy chain variable regions of the above-mentioned anti-GPC3 humanized antibody, and finally obtains four anti-GPC3 humanized antibodies, The specific combinations are shown in Table 9.
- the anti-GPC3 humanized antibody scFv-human IgG1 Fc (scFv-hFc) was constructed to form the form, and the Fc region sequence is shown in the sequence of SEQ ID NO: 1 Nos. 244-475.
- Example 1(A) ELISA detection and data analysis were carried out according to the method of Example 1(A).
- the OD450nm value was read with an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer).
- the results of the binding activity of the anti-GPC3 humanized antibody to human GPC3 protein are shown in Figure 6 and Table 11.
- Human GPC3 protein binds well , where the IgG control is hIgG1, and the data in the table are OD 450nm values.
- Table 11 ELISA detects the binding reaction of anti-GPC3 humanized antibody to human GPC3 protein
- the desired cells were expanded to logarithmic growth phase in T-75 cell culture flasks, the medium was aspirated, washed twice with PBS buffer, the cells were trypsinized, then the digestion was terminated with complete medium, and the cells were pipetted to single-cell suspension. After cell counting, centrifuge, resuspend the cell pellet with FACS buffer (PBS+2% fetal bovine serum) to 2x10 6 cells per ml, add 50 ⁇ l per well to a 96-well FACS reaction plate, add anti-GPC3 humanized Antibody test samples were 50 ⁇ l per well and incubated at 4°C for 1 hour.
- FACS buffer PBS+2% fetal bovine serum
- Table 12 and Figures 7A and 7B show that all four anti-GPC3 humanized antibodies can bind to CHO-K1-human GPC3 cells, but not to CHOK1 cells ;
- Table 13 and Figures 8A and 8B show that all four anti-GPC3 humanized antibodies can bind to CHO-K1-human GPC3 cells. Binds to HepG2 cells, and neither binds to A431 cells .
- Monkey GPC3-His protein purchased from Acro, product number: GP3-C5225
- mouse GPC3-his protein purchased from Sino Biological, product number: 50989-M08B
- the ELISA results of anti-GPC3 humanized antibodies and murine GPC3 protein are shown in Figure 9 and Table 14. The results show that none of the four anti-GPC3 humanized antibodies binds to murine GPC3 protein .
- the IgG control is hIgG1
- the data in the table are OD 450nm values.
- the ELISA results of the anti-GPC3 humanized antibodies and the monkey GPC3 protein are shown in Figure 10 and Table 15. The results show that the four anti-GPC3 humanized antibodies bind well to the monkey GPC3 protein .
- Table 15 ELISA detects the binding reaction of anti-GPC3 humanized antibody to monkey GPC3 protein
- the HEK293T-monkey GPC3 cells were subjected to FACS detection and data analysis according to the method of Example 3(B). The analysis results are shown in Table 16 and Figures 11A and 11B. All four anti-GPC3 humanized antibodies bind to HEK293T-monkey-GPC3 cells, but not to HEK293T cells .
- Anti-GPC3 humanized antibodies were captured using a Protein A chip (GE Helthcare; 29-127-558).
- Sample and running buffer were HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
- the flow-through cell was set to 25 °C.
- the sample block was set to 16°C. Both were pretreated with running buffer.
- the antibody to be tested was first captured with a Protein A chip, and then a single concentration of GPC3 antigen protein was injected to record the binding and dissociation process of the antibody and the antigen protein.
- Glycine pH1.5 (GE Helthcare; BR-1003- 54) Complete chip regeneration.
- Binding was measured by injecting various concentrations of human GPC3-His in solution for 240 sec with a flow rate of 30 ⁇ L/min, starting at 200 nM (see detailed results for actual concentrations tested), diluted 1:1 for a total of 5 concentrations.
- the dissociation phase was monitored for up to 600 seconds and triggered by switching from sample solution to running buffer.
- the binding rate (K a ), dissociation rate (K d ) and binding affinity (KD) of the anti-GPC3 humanized antibody to human GPC3 protein are shown in the table, in which the antibody Y035 was used as a control. As shown in Table 17, the affinities of the four anti-GPC3 humanized antibodies to human GPC3 protein were all above 1E-8M .
- the affinity test of the anti-GPC3 humanized antibody and monkey GPC3-His protein was carried out according to the method of Example 5(A), wherein the antibody Y035 was used as a control. As shown in Table 18, the affinity of the four anti-GPC3 humanized antibodies to the monkey GPC3 protein was all above 1E-8M, and the affinity of SIM5 was better than 1E-9M, reaching 5.41E-10M .
- the mature GPC3 protein has a soluble amino-terminal (N-terminal) peptide of about 40 kD and a membrane-bound carboxyl-terminal (C-terminal) peptide of about 30 kD that can enter the blood.
- the Y035 antibody recognizes the C-terminal region of GPC3 protein close to the cell membrane (proximal end), and the T2-23 antibody recognizes the non-membrane-proximal region.
- the polypeptide GC3pep (membrane-near end) coated with human GPC3 was subjected to the proximity of the anti-GPC3 humanized antibody.
- Membrane end binding identification as shown in Figure 12 and Table 19, all four anti-GPC3 humanized antibodies can recognize GC3pep, belonging to the antibodies that recognize the near-membrane end epitope.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- General Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Food Science & Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Pathology (AREA)
- Veterinary Medicine (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- Hospice & Palliative Care (AREA)
Abstract
Description
抗体名称 | VL | VH |
S001-SIM2 | GPC3.VL1 | GPC3.VH2 |
S001-SIM3 | GPC3.VL2a | GPC3.VH1 |
S001-SIM4 | GPC3.VL2a | GPC3.VH2 |
S001-SIM5 | GPC3.VL2a | GPC3.VH5 |
抗体名称 | Ka(1/Ms) | Kd(1/s) | KD(M) |
S001-SIM2 | 1.92E+05 | 3.91E-04 | 2.04E-09 |
S001-SIM3 | 1.94E+05 | 4.11E-04 | 2.12E-09 |
S001-SIM4 | 1.87E+05 | 3.67E-04 | 1.96E-09 |
S001-SIM5 | 2.11E+05 | 2.25E-04 | 1.07E-09 |
Y035 | 1.55E+05 | 2.73E-04 | 1.76E-09 |
抗体名称 | Ka(1/Ms) | Kd(1/s) | KD(M) |
S001-SIM2 | 2.76E+05 | 4.07E-04 | 1.47E-09 |
S001-SIM3 | 3.38E+05 | 3.83E-04 | 1.13E-09 |
S001-SIM4 | 3.08E+05 | 3.66E-04 | 1.19E-09 |
S001-SIM5 | 3.75E+05 | 2.03E-04 | 5.41E-10 |
Y035 | 2.47E+05 | 3.13E-04 | 1.27E-09 |
Claims (29)
- 一种特异性结合GPC3的抗体或抗原结合片段,其包含人源化的重链可变区和/或人源化的轻链可变区,所述重链可变区包含人类来源的重链框架区、包含SEQ ID NO:15的氨基酸序列的互补决定区(CDR)1、包含SEQ ID NO:16的氨基酸序列的CDR2、和包含SEQ ID NO:17的氨基酸序列的CDR3;所述轻链可变区包含人类来源的轻链框架区、包含SEQ ID NO:18、21的氨基酸序列的互补决定区(CDR)1、包含SEQ ID NO:19的氨基酸序列的CDR2、和包含SEQ ID NO:20的氨基酸序列的CDR3。
- 权利要求1所述的抗体或抗原结合片段,其特征在于,所述重链框架区包含SEQ ID NO:11所示IGHV1-3*01的框架区HFR1、HFR2和HFR3,以及SEQ ID NO:12所示IGHJ1*01的框架区HFR4;所述轻链框架区包含SEQ ID NO:9所示IGKV2-40*01的框架区LFR1、LFR2和LFR3,以及SEQ ID NO:10所示IGKJ2*01的框架区LFR4,其中,所述框架区根据Kabat编号系统确定。
- 权利要求2所述的抗体或抗原结合片段,其特征在于,根据Kabat编号系统编号,所述重链框架区包含选自第1、44、69、71、73、93位上的氨基酸残基突变,优选地,所述突变包含:Q1E、R71A和A93T;Q1E、I69L、R71A和A93T;或Q1E、R44G、I69L、R71A、T73K和A93T。
- 权利要求2或3所述的抗体或抗原结合片段,其特征在于,根据Kabat编号系统编号,所述轻链框架区包含至多一个氨基酸残基突变;优选地,所述突变为选自第2位上的氨基酸残基突变;优选地,所述突变为I2V。
- 权利要求1-4任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包含:(1)SEQ ID NO:4-6任一项所示的重链可变区,或,具有与SEQ ID NO:4-6任一项所示序列相比具有至少80、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性的序列;或,具有与SEQ ID NO:4-6任一项所示序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12 个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列;所述突变可选自插入、缺失和/或替换,所述替换优选为保守氨基酸的替换;和/或,(2)SEQ ID NO:7-8、13-14任一项所示的轻链可变区,或,具有与SEQ ID NO:7-8、13-14任一项所示序列相比具有至少80、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性的序列;或,具有与SEQ ID NO:7-8、13-14任一项所示序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列;所述突变可选自插入、缺失和/或替换,所述替换优选为保守氨基酸的替换。
- 权利要求5所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包含:(1)具有SEQ ID NO:4、5、6所示序列的重链可变区,和具有SEQ ID NO:14所示序列的轻链可变区;或,(2)具有SEQ ID NO:5所示序列的重链可变区,和具有SEQ ID NO:7所示序列的轻链可变区;或,(3)分别具有与上述(1)至(2)所示序列具有至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或更高一致性的序列的重链可变区和轻链可变区。
- 权利要求1-6任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包括与所述CDR1、CDR2和/或CDR3相比具有至少80、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性的序列;或与所述CDR1、CDR2和/或CDR3相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列,所述替换优选为保守氨基酸的替换。
- 权利要求1-7任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段特异性结合人和/或猴GPC3蛋白;优选地,与人和/或猴GPC3的解离常数(KD)不大于1.00E-7 M、1.00E-8 M、2.00E-8 M、3.00E-8 M、4.00E-8 M、5.00E-8 M、6.00E-8 M、7.00E-8 M、8.00E-8 M、9.00E-8 M、1.00E-9 M、2.00E-9 M、3.00E-9 M、4.00E-9 M、5.00E-9 M、6.00E-9 M、7.00E-9 M、8.00E-9 M、9.00E-9 M或6.00E-10 M。
- 根据权利要求1-8任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段选自全长抗体、VH单域结构抗体、Fab片段、Fab'片段、F(ab)'2片段、Fd片段、Fv片段、互补决定区(CDR)片段、单链可变片段(scFv)、scFV2、二硫键稳定的可变片段(dsFv)、结构域抗体、二价单链抗体、单链噬菌体抗体、双特异双链抗体、三链抗体、四链抗体或抗体最小识别单位。
- 一种免疫缀合物,其特征在于,所述免疫缀合物包含权利要求1-9任一项所述的抗体或抗原结合片段和效应分子;优选地,所述效应分子与所述抗体或抗原结合片段连接。
- 根据权利要求10所述的免疫缀合物,其特征在于,所述效应分子包括治疗剂或标记物;优选地,所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂。
- 根据权利要求10或11所述的免疫缀合物,其特征在于,所述免疫缀合物还包括用于将所述效应分子与所述抗体缀合的接头,所述接头包括但不限于腙、硫醚、酯、二硫化物和含肽的接头。
- 一种嵌合抗原受体(CAR),其特征在于,所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域,所述细胞外抗原结合结构域包含权利要求1-9任一项所述抗体或抗原结合片段。
- 一种免疫活性细胞,其特征在于,所述免疫活性细胞表达权利要求13所述的嵌合抗原受体或包含编码权利要求13所述嵌合抗原受体的核酸分子;优选地,所述免疫活性细胞选自:T细胞,NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞,所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞。
- 一种多特异性分子,其特征在于,所述多特异性分子包含权利要求1-9中任一项所述的抗体或抗原结合片段;优选地,所述多特异性分子 进一步包含特异性结合GPC3以外的抗原或结合与权利要求1-9任一项所述抗体或抗原结合片段不同的GPC3表位的抗体或抗原结合片段。
- 根据权利要求15所述的多特异性分子,其特征在于,所述GPC3以外的抗原为T细胞、B细胞、自然杀伤细胞、树突状细胞、巨噬细胞、单核细胞或嗜中性细胞表面上的抗原;优选地,所述GPC3以外的抗原选自:CD3、CD3γ、CD3δ、CD3ε、CD3ζ、CD16、CD16A、CD32B、PD-1、PD-2、PD-L1、VEGF、NKG2D、CD19、CD20、CD40、CD47、4-1BB、CD137、EGFR、EGFRvIII、TNF-alpha、CD33、HER2、HER3、HAS、CD5、CD27、EphA2、EpCAM、MUC1、MUC16、CEA、Claudin18.2、叶酸受体、Claudin6、WT1、NY-ESO-1、MAGE3、ASGPR1或CDH16。
- 根据权利要求15或16所述的多特异性分子,其特征在于,所述多特异性分子为串联scFv、双功能抗体(Db)、单链双功能抗体(scDb)、双重亲和力再靶向(DART)抗体、F(ab')2、双重可变域(DVD)抗体、臼包杵(KiH)抗体、对接及锁定(DNL)抗体、化学交联抗体、杂多聚抗体或异结合物抗体。
- 一种分离的核酸分子,其特征在于,所述核酸分子编码权利要求1-9任一项所述的抗体或抗原结合片段、权利要求13所述的嵌合抗原受体、或权利要求15-17任一项所述的多特异性分子。
- 一种载体,其特征在于,所述表达载体包含权利要求18所述的核酸分子。
- 一种宿主细胞,其特征在于,所述宿主细胞包含权利要求18所述的核酸分子或权利要求19所述的表达载体,优选地,所述宿主细胞为原核细胞或真核细胞,包括细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293细胞系)。
- 一种制备权利要求1-9任一项所述的抗体或抗原结合片段、或权利要求15-17任一项所述的多特异性分子的方法,其特征在于,所述方法包括培养权利要求20所述的细胞,以及分离所述细胞表达的抗体或抗原结合片段,或分离所述细胞表达的多特异性分子。
- 一种制备权利要求14所述的免疫活性细胞的方法,其特征在于, 所述方法包括:将包含编码权利要求13所述嵌合抗原受体的核酸片段导入所述免疫活性细胞,可选地,所述方法还包括启动所述免疫活性细胞表达权利要求13所述嵌合抗原受体。
- 一种药物组合物,其特征在于,所述药物组合物包括治疗有效量的一种或组合的:权利要求1-9所述的抗体或抗原结合片段;或权利要求10-12所述的免疫缀合物;或权利要求14所述的免疫活性细胞;或权利要求15-17所述的多特异性分子;或权利要求18所述的核酸分子;或权利要求19所述的表达载体;或根据权利要求21-22任一项所述方法制备获得的产品,以及药学上可接受的载体。
- 权利要求1-9所述的抗体或抗原结合片段;或权利要求10-12所述的免疫缀合物;或权利要求14所述的免疫活性细胞;或权利要求15-17所述的多特异性分子;或权利要求18所述的核酸分子;或权利要求19所述的表达载体;或根据权利要求21-22任一项所述方法制备获得的产品或权利要求23所述的药物组合物在制备治疗GPC3介导的肿瘤的药物中的用途;优选地,所述肿瘤选自肝细胞癌、黑色素瘤、卵巢透明细胞癌、肝胚细胞瘤、成神经细胞瘤、肾母细胞瘤、小细胞肺癌、肺腺癌、胃癌、结肠癌、直肠癌、子宫颈癌、乳腺癌、卵巢癌,皮肤癌、淋巴癌、前列腺癌、胰腺癌、肾癌、食道癌、甲状腺癌、睾丸癌、膀胱癌、支气管癌、鼻咽癌、头颈癌、子宫内膜癌、脑癌、骨癌、白血病、恶性间皮瘤、脂肪肉瘤等疾病;优选为肝细胞癌。
- 一种治疗患有GPC3介导的肿瘤的受试者的方法,其特征在于,给予所述受试者治疗有效量的权利要求1-9所述的抗体或抗原结合片段;或权利要求10-12所述的免疫缀合物;或权利要求14所述的免疫活性细胞;或权利要求15-17所述的多特异性分子;或权利要求18所述的核酸分子;或权利要求19所述的表达载体;或根据权利要求21-22任一项所述方法制备获得的产品或权利要求23所述的药物组合物;优选地,所述肿瘤选自肝细胞癌、黑色素瘤、卵巢透明细胞癌、肝胚细胞瘤、成神经细胞瘤、肾母细胞瘤、小细胞肺癌、肺腺癌、胃癌、结肠癌、直肠癌、子宫颈癌、乳腺癌、卵巢癌,皮肤癌、淋巴癌、前列腺癌、胰腺癌、肾癌、食 道癌、甲状腺癌、睾丸癌、膀胱癌、支气管癌、鼻咽癌、头颈癌、子宫内膜癌、脑癌、骨癌、白血病、恶性间皮瘤、脂肪肉瘤等疾病;优选为肝细胞癌。
- 权利要求1-9所述的抗体或抗原结合片段;或权利要求10-12所述的免疫缀合物;或权利要求14所述的免疫活性细胞;或权利要求15-17所述的多特异性分子;或权利要求18所述的核酸分子;或权利要求19所述的表达载体;或根据权利要求21-22任一项所述方法制备获得的产品或权利要求23所述的药物组合物,其特征在于,用于治疗GPC3阳性肿瘤或癌症;优选地,所述肿瘤选自肝细胞癌、黑色素瘤、卵巢透明细胞癌、肝胚细胞瘤、成神经细胞瘤、肾母细胞瘤、小细胞肺癌、肺腺癌、胃癌、结肠癌、直肠癌、子宫颈癌、乳腺癌、卵巢癌,皮肤癌、淋巴癌、前列腺癌、胰腺癌、肾癌、食道癌、甲状腺癌、睾丸癌、膀胱癌、支气管癌、鼻咽癌、头颈癌、子宫内膜癌、脑癌、骨癌、白血病、恶性间皮瘤、脂肪肉瘤等疾病;优选为肝细胞癌。
- 一种试剂盒,其包含权利要求1-9所述的抗体或抗原结合片段;或权利要求10-12所述的免疫缀合物;或权利要求14所述的免疫活性细胞;或权利要求15-17所述的多特异性分子;或权利要求18所述的核酸分子;或权利要求19所述的表达载体;或根据权利要求21-22任一项所述方法制备获得的产品或权利要求23所述的药物组合物。
- 权利要求1-9所述的抗体或抗原结合片段在制备检测或诊断GPC3高表达肿瘤的试剂中的用途。
- 一种检测生物学样品中GPC3表达的方法,其特征在于,使来自受试者的样品与权利要求1-9所述的抗体或抗原结合片段接触,并检测所述抗体或抗原结合片段与所述样品的结合。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202280013317.1A CN117062834A (zh) | 2021-02-10 | 2022-02-09 | Gpc3人源化抗体及其应用 |
US18/275,901 US20240301085A1 (en) | 2021-02-10 | 2022-02-09 | Humanized gpc3 antibody and application thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110182284 | 2021-02-10 | ||
CN202110182284.0 | 2021-02-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022171100A1 true WO2022171100A1 (zh) | 2022-08-18 |
Family
ID=82838284
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2022/075588 WO2022171100A1 (zh) | 2021-02-10 | 2022-02-09 | Gpc3人源化抗体及其应用 |
Country Status (3)
Country | Link |
---|---|
US (1) | US20240301085A1 (zh) |
CN (1) | CN117062834A (zh) |
WO (1) | WO2022171100A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024114404A1 (zh) * | 2022-11-28 | 2024-06-06 | 成都科伦精准生物科技有限公司 | 特异性结合gpc3的嵌合抗原受体及其应用 |
WO2024114410A1 (zh) * | 2022-11-28 | 2024-06-06 | 四川科伦博泰生物医药股份有限公司 | 靶向gpc3的抗体及其用途 |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101068836A (zh) * | 2004-10-26 | 2007-11-07 | 中外制药株式会社 | 糖链改变的抗磷脂酰肌醇聚糖3抗体 |
CN102046200A (zh) * | 2008-04-04 | 2011-05-04 | 中外制药株式会社 | 肝癌治疗剂 |
CN104871003A (zh) * | 2012-12-21 | 2015-08-26 | 中外制药株式会社 | 用于向gpc3靶向治疗剂疗法有效的患者施予的gpc3靶向治疗剂 |
CN106459959A (zh) * | 2014-05-08 | 2017-02-22 | 中外制药株式会社 | 对gpc3靶向治疗剂疗法有效的患者施用的gpc3靶向治疗剂 |
CN108164600A (zh) * | 2016-12-07 | 2018-06-15 | 上海吉倍生物技术有限公司 | 一种抗gpc3抗体及其制备方法和用途 |
US20190046659A1 (en) * | 2015-08-03 | 2019-02-14 | Carsgen Therapeutics Ltd | Antibody against glypican-3 and application thereof |
CN109988240A (zh) * | 2017-12-29 | 2019-07-09 | 安源生物科技(上海)有限公司 | 抗gpc-3抗体及其用途 |
WO2020017479A1 (ja) * | 2018-07-17 | 2020-01-23 | ノイルイミューン・バイオテック株式会社 | 抗gpc3一本鎖抗体を含むcar |
CN112390886A (zh) * | 2019-08-16 | 2021-02-23 | 上海原能细胞医学技术有限公司 | 一种分离的抗原结合蛋白及其用途 |
-
2022
- 2022-02-09 WO PCT/CN2022/075588 patent/WO2022171100A1/zh active Application Filing
- 2022-02-09 US US18/275,901 patent/US20240301085A1/en active Pending
- 2022-02-09 CN CN202280013317.1A patent/CN117062834A/zh active Pending
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101068836A (zh) * | 2004-10-26 | 2007-11-07 | 中外制药株式会社 | 糖链改变的抗磷脂酰肌醇聚糖3抗体 |
CN102046200A (zh) * | 2008-04-04 | 2011-05-04 | 中外制药株式会社 | 肝癌治疗剂 |
CN104871003A (zh) * | 2012-12-21 | 2015-08-26 | 中外制药株式会社 | 用于向gpc3靶向治疗剂疗法有效的患者施予的gpc3靶向治疗剂 |
CN106459959A (zh) * | 2014-05-08 | 2017-02-22 | 中外制药株式会社 | 对gpc3靶向治疗剂疗法有效的患者施用的gpc3靶向治疗剂 |
US20190046659A1 (en) * | 2015-08-03 | 2019-02-14 | Carsgen Therapeutics Ltd | Antibody against glypican-3 and application thereof |
CN108164600A (zh) * | 2016-12-07 | 2018-06-15 | 上海吉倍生物技术有限公司 | 一种抗gpc3抗体及其制备方法和用途 |
CN109988240A (zh) * | 2017-12-29 | 2019-07-09 | 安源生物科技(上海)有限公司 | 抗gpc-3抗体及其用途 |
WO2020017479A1 (ja) * | 2018-07-17 | 2020-01-23 | ノイルイミューン・バイオテック株式会社 | 抗gpc3一本鎖抗体を含むcar |
CN112390886A (zh) * | 2019-08-16 | 2021-02-23 | 上海原能细胞医学技术有限公司 | 一种分离的抗原结合蛋白及其用途 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024114404A1 (zh) * | 2022-11-28 | 2024-06-06 | 成都科伦精准生物科技有限公司 | 特异性结合gpc3的嵌合抗原受体及其应用 |
WO2024114410A1 (zh) * | 2022-11-28 | 2024-06-06 | 四川科伦博泰生物医药股份有限公司 | 靶向gpc3的抗体及其用途 |
Also Published As
Publication number | Publication date |
---|---|
US20240301085A1 (en) | 2024-09-12 |
CN117062834A (zh) | 2023-11-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11753473B2 (en) | Anti-PD-L1 antibodies | |
WO2020135201A1 (zh) | 一种抗体及其用途 | |
WO2019184909A1 (zh) | 新型抗体分子、其制备方法及其用途 | |
CN112969476B (zh) | 多特异性蛋白分子 | |
TW201833141A (zh) | 抗b7-h3抗體、其抗原結合片段及其醫藥用途 | |
WO2020168555A1 (zh) | Cd3抗原结合片段及其应用 | |
CN112969719A (zh) | 双功能融合蛋白及其医药用途 | |
WO2022166876A1 (zh) | 特异性识别磷脂酰肌醇蛋白聚糖3的单克隆抗体及其应用 | |
WO2022171100A1 (zh) | Gpc3人源化抗体及其应用 | |
WO2022135536A1 (zh) | Cd3人源化抗体及其应用 | |
TWI821474B (zh) | Cd3抗體及其藥物用途 | |
CN110790839A (zh) | 抗pd-1抗体、其抗原结合片段及医药用途 | |
KR20230028708A (ko) | 항-b7-h4/항-4-1bb 이중특이적 항체 및 이의 용도 | |
WO2022121941A1 (zh) | 抗人msln的抗体及其用途 | |
WO2022184162A1 (zh) | 针对NKp46的抗体及其应用 | |
EP4269442A1 (en) | Mesothelin binding molecule and application thereof | |
JP2023540436A (ja) | Pd-l1およびtgf-βを標的とする二機能性分子 | |
WO2023041065A1 (zh) | 一种靶向人ceacam5/6的抗体、制备方法和应用 | |
WO2024061223A1 (zh) | 抗体及其在抗肿瘤中的应用 | |
WO2023155926A1 (en) | Tumor antigen-dependent cd40 agonist antibodies | |
WO2024078558A1 (zh) | 抗cd100抗体及其用途 | |
EP4410834A1 (en) | Ctla-4 binding molecule and use thereof | |
WO2022110922A1 (zh) | 抗SIRPα抗体或其抗原结合片段及应用 | |
TWI751110B (zh) | Aml抗原及其用途 | |
CN115572330A (zh) | 一种特异性抗体及其制备方法和应用 |
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: 22752260 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 18275901 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 202280013317.1 Country of ref document: CN |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 22752260 Country of ref document: EP Kind code of ref document: A1 |