WO2024067612A1 - Anticorps anti-claudine 18.2 - Google Patents
Anticorps anti-claudine 18.2 Download PDFInfo
- Publication number
- WO2024067612A1 WO2024067612A1 PCT/CN2023/121643 CN2023121643W WO2024067612A1 WO 2024067612 A1 WO2024067612 A1 WO 2024067612A1 CN 2023121643 W CN2023121643 W CN 2023121643W WO 2024067612 A1 WO2024067612 A1 WO 2024067612A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- antigen
- binding fragment
- cells
- seq
- Prior art date
Links
- 230000027455 binding Effects 0.000 claims abstract description 99
- 239000000427 antigen Substances 0.000 claims abstract description 76
- 102000036639 antigens Human genes 0.000 claims abstract description 76
- 108091007433 antigens Proteins 0.000 claims abstract description 76
- 239000012634 fragment Substances 0.000 claims abstract description 59
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 28
- 239000013598 vector Substances 0.000 claims abstract description 25
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 16
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 16
- 239000002157 polynucleotide Substances 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 156
- 150000001413 amino acids Chemical class 0.000 claims description 47
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 13
- 201000011510 cancer Diseases 0.000 claims description 10
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 8
- 206010017758 gastric cancer Diseases 0.000 claims description 8
- 201000011549 stomach cancer Diseases 0.000 claims description 8
- 210000003236 esophagogastric junction Anatomy 0.000 claims description 7
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 4
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 4
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 4
- 208000009956 adenocarcinoma Diseases 0.000 claims description 4
- 201000004101 esophageal 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
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 3
- 230000002496 gastric effect Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 101000827703 Homo sapiens Polyphosphoinositide phosphatase Proteins 0.000 claims 2
- 102100023591 Polyphosphoinositide phosphatase Human genes 0.000 claims 2
- 239000008194 pharmaceutical composition Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 46
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 31
- 229950007157 zolbetuximab Drugs 0.000 description 25
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 18
- 230000035772 mutation Effects 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 102000039446 nucleic acids Human genes 0.000 description 13
- 108020004707 nucleic acids Proteins 0.000 description 13
- 150000007523 nucleic acids Chemical class 0.000 description 13
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 12
- 210000000822 natural killer cell Anatomy 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 11
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 11
- 238000001514 detection method Methods 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 210000004408 hybridoma Anatomy 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 230000005888 antibody-dependent cellular phagocytosis Effects 0.000 description 9
- 229920001184 polypeptide Polymers 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 210000004881 tumor cell Anatomy 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 238000000684 flow cytometry Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 6
- 230000012202 endocytosis Effects 0.000 description 6
- 230000002147 killing effect Effects 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 229920002477 rna polymer Polymers 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 102000002038 Claudin-18 Human genes 0.000 description 4
- 108050009324 Claudin-18 Proteins 0.000 description 4
- 206010057249 Phagocytosis Diseases 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 230000000242 pagocytic effect Effects 0.000 description 4
- 230000008782 phagocytosis Effects 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 3
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102100029193 Low affinity immunoglobulin gamma Fc region receptor III-A Human genes 0.000 description 3
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000002121 endocytic effect Effects 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 230000005909 tumor killing Effects 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- 102100021266 Alpha-(1,6)-fucosyltransferase Human genes 0.000 description 2
- 102000002029 Claudin Human genes 0.000 description 2
- 108050009302 Claudin Proteins 0.000 description 2
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 2
- 101000819490 Homo sapiens Alpha-(1,6)-fucosyltransferase Proteins 0.000 description 2
- 238000005588 Kraus reaction Methods 0.000 description 2
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 2
- 101710099301 Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000006317 isomerization reaction Methods 0.000 description 2
- 238000000111 isothermal titration calorimetry Methods 0.000 description 2
- 230000010534 mechanism of action Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 102000014447 Complement C1q Human genes 0.000 description 1
- 108010078043 Complement C1q Proteins 0.000 description 1
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 102220556549 Delta and Notch-like epidermal growth factor-related receptor_Q47M_mutation Human genes 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102220593368 Exophilin-5_Q29M_mutation Human genes 0.000 description 1
- 241000126130 Ganymedes Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 101000839781 Homo sapiens Immunoglobulin heavy variable 4-59 Proteins 0.000 description 1
- 101001121408 Homo sapiens L-amino-acid oxidase Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- 102100028405 Immunoglobulin heavy variable 4-59 Human genes 0.000 description 1
- 102100026388 L-amino-acid oxidase Human genes 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
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 102220484769 Olfactory receptor 5A1_A42S_mutation Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102220536901 PRKC apoptosis WT1 regulator protein_L69I_mutation Human genes 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100012902 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FIG2 gene Proteins 0.000 description 1
- 101100233916 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) KAR5 gene Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102220630036 Stress-responsive DNAJB4-interacting membrane protein 1_N45Q_mutation Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 108010076089 accutase Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000009824 affinity maturation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000611 antibody drug conjugate Substances 0.000 description 1
- 229940049595 antibody-drug conjugate Drugs 0.000 description 1
- 230000009227 antibody-mediated cytotoxicity Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000005975 antitumor immune response Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 102220344885 c.67A>T Human genes 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 125000003712 glycosamine group Chemical group 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 210000000723 mammalian artificial chromosome Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 102220059322 rs786203566 Human genes 0.000 description 1
- 102220095187 rs876658411 Human genes 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 210000001578 tight junction Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- 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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- 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
Definitions
- the present invention belongs to the field of tumor immunity, and more specifically, the present invention relates to an antibody against claudin 18.2 and its application in the field of tumor immunity.
- Monoclonal antibody-based immunotherapy is now a major component of cancer treatment, alongside surgery, radiation, and chemotherapy.
- Monoclonal antibodies have a variety of clinically relevant mechanisms of action, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and direct receptor or ligand blockade.
- ADCC antibody-dependent cellular cytotoxicity
- CDC complement-dependent cytotoxicity
- ADCP antibody-dependent cellular phagocytosis
- direct receptor or ligand blockade In addition, antibodies can simultaneously promote the induction of long-lasting antitumor immune responses (Welschof & Krauss, 2003).
- the multifaceted nature of antibodies as therapeutic platforms has led to the development of new cancer treatment strategies that will have a significant impact on cancer treatment.
- Gastric cancer is a malignant tumor originating from the mucosal epithelium and is one of the most common cancers in the world. According to statistics from the World Health Organization's cancer control program, 7 million people die of cancer each year worldwide, of which 700,000 die from gastric cancer. Compared with conventional gastric cancer treatment options, such as surgery and chemotherapy, tumor immunotherapy has the characteristics of high specificity and low side effects, and has a more far-reaching application prospect (Yasui et al., 2011).
- Claudin-18 (CLDN18) is an intrinsic membrane protein located in the tight junctions of epithelium and endothelium. It has four transmembrane hydrophobic regions and two extracellular loops, with a molecular weight of about 27.9KD (Krause et al., 2008). Claudin-18 exists in two different splicing forms: Claudin-18.1 (CLDN18.1) and Claudin-18.2 (CLDN18.2).
- Claudin-18.1 is selectively expressed in normal lung or gastric cells
- Claudin-18.2 is normally only expressed in differentiated gastric epithelial short-cycle cells of the stomach, but is highly expressed in a variety of tumor tissues, such as gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, breast cancer, etc.
- GEJ gastroesophageal junction
- the differential expression of Claudin-18.2 in normal cells and tumor cells makes it an attractive target for cancer immunotherapy.
- the first drug developed for the Claudin18.2 target is Zolbetuximab (IMAB362) from GANYMED Pharmaceuticals, a chimeric antibody that has not been humanized and has entered the clinical stage (Sahin et al., 2018; Woll et al., 2014). Antibodies that are specific to these cancers are of great significance for the treatment and detection of the above cancers.
- the technical problem to be solved by the present invention is to obtain an antibody that specifically binds to CLDN18.2 but not to CLDN18.1, has high affinity, low immunogenicity, and has good clinical prospects.
- the present invention provides an anti-CLDN18.2 antibody or antibody fragment, and based on the antibody or the fragment thereof, provides its use.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, comprising:
- the anti-CLDN18.2 antibodies of the present invention may be humanized antibodies.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein: the light chain variable region comprises one or more amino acids selected from the group consisting of 23L, 54I, 59A and 116S; and/or the heavy chain variable region comprises one or more amino acids selected from the group consisting of 181W, 201N, 218K, 245G/S and 250F, wherein the amino acids are numbered as shown in FIG. 4 .
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein: the light chain variable region comprises amino acids corresponding to 23L, 54I, 59A, 116S, 23L/59A, 23L/116S, 59A/116S or 23L/59A/116S; and/or the heavy chain variable region comprises amino acids corresponding to 181W, 201N, 218K, 245G, 245S, 250F, 181W/250F or 201N/245S, wherein the amino acids are numbered as shown in FIG. 4 .
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein: the light chain variable region comprises amino acids corresponding to 23L, 59A, 23L/59A, 23L/116S, 59A/116S or 23L/59A/116S; and/or the heavy chain variable region comprises amino acids corresponding to 201N, 218K, 245G, 245S, 250F, or 201N/245S, wherein the amino acids are numbered as shown in FIG. 4 .
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the light chain variable region comprises amino acids corresponding to 23L and 116S, and the heavy chain variable region comprises amino acids corresponding to 218K, wherein the amino acids are numbered as shown in FIG. 4 .
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein: the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 16, 35-40, 43 and 46; and/or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 14, 31-34, 41, 42, 44 and 45.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the antibody comprises: a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 22 and 26; and/or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 20 and 24.
- the antigen binding fragment is selected from the group consisting of: Fab, Fab', F(ab') 2 , diabody, Fd, and Fd' fragments.
- a polynucleotide encoding the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention is provided.
- the polynucleotide comprises a nucleotide sequence selected from SEQ ID NO:11, 12, 15, 17, 21, 23, 25 and 27.
- the present invention provides a vector comprising the polynucleotide of the second aspect of the present invention, and further provides a host cell comprising the polynucleotide of the second aspect of the present invention, or comprising the vector of the present invention.
- the host cell is a eukaryotic cell or a prokaryotic cell.
- the host cell is selected from CHO cells, COS cells, HeLa cells, HEK cells, such as HEK 293 cells.
- the present invention provides a method for producing the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention, comprising the steps of: culturing the cell according to the present invention; and recovering the anti-CLDN18.2 antibody or antigen-binding fragment thereof from the culture.
- the present invention provides a composition for diagnosing, preventing and/or treating a tumor associated with CLDN18.2, comprising the anti-CLDN18.2 antibody or antigen-binding fragment thereof, polynucleotide or vector according to the present invention.
- the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, polynucleotide, vector or composition of the present invention for use in the preparation of a method for diagnosis, prevention and/or Use in a composition for treating tumors associated with CLDN18.2.
- the tumor is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, and breast cancer.
- GEJ gastroesophageal junction
- the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention shows high affinity and specificity for CLDN18.2, and can effectively bind to target cells with relatively low density of CLDN18.2 expression abundance. Furthermore, the humanized anti-CLDN18.2 antibody and antigen-binding fragment thereof of the present invention also show significantly enhanced overall immune activity, including endocytosis activity, CDC activity, ADCC activity and ADCP phagocytosis.
- antibodies e.g., monoclonal antibodies
- antigen-binding fragments thereof that specifically bind to CLDN18.2.
- monoclonal anti-CLDN18.2 antibodies that specifically bind to CLDN18.2, wherein the anti-CLDN18.2 antibodies include variants of parent antibodies.
- antibodies that specifically bind to CLDN18.2 e.g., human CLDN18.2.
- CLDN18.2 refers to any CLDN18.2 membrane protein known to those skilled in the art.
- the CLDN18.2 may be from a mammal, such as CLDN18.2 may be from a human or a cynomolgus monkey.
- the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.
- amino acid residues in the amino acid sequence are represented in the form of numbers and single-letter abbreviations, wherein the numbers represent the position of the amino acid residue in the amino acid sequence, the letters on the left side of the numbers represent the amino acids before substitution, and the letters on the right side of the numbers represent the different amino acids after substitution.
- amino acid substitution "M23L” means that the methionine at position 23 of the amino acid sequence is replaced by leucine. In the absence of letters on the left, it represents the specific amino acid at the indicated amino acid sequence position.
- amino acid corresponding to "245G/S" means that the amino acid at position 245 of the amino acid sequence is glycine or serine.
- variable domains of antibodies refers to certain parts of related molecules that have extensive sequence differences between antibodies and are used for specific recognition and binding of a particular antibody to its specific target.
- the variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located in the variable domains of both the light and heavy chains.
- CDRs complementarity determining regions
- hypervariable regions which are located in the variable domains of both the light and heavy chains.
- the more conserved portions of the variable domains are called framework (FR) regions or framework sequences.
- Each variable domain of native heavy and light chains includes four framework regions, which mainly adopt a ⁇ -sheet configuration, which are connected by three CDRs to form loops that contribute to the formation of the antibody target binding site (epitope or determinant) (see Kabat et al. (Kabat, 1991)).
- the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
- a CDR can have the ability to specifically bind to a cognate epitope.
- an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody, but contains at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody combining sites) of the antibody that binds to the antigen and thus retains the binding specificity to and at least part of the specific binding ability of the full-length antibody.
- an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
- Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.
- Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, diabodies, Fd and Fd' fragments and other fragments, including modified fragments (Welschof & Krauss, 2003).
- the fragments may include multiple chains linked together, for example, by disulfide bonds and/or by peptide linkers.
- Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
- Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains an antibody that immunospecifically binds (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M -1 ) to an antigen.
- “Functional fragments” or “analogs of anti-CLDN18.2 antibodies” are fragments or analogs that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction.
- functional fragments are generally synonymous with "antibody fragments”, and in the case of antibodies, can refer to fragments that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab') 2 , etc.
- “Fv” fragments are composed of a dimer (VH-VL dimer) formed by non-covalent binding of the variable domain of a heavy chain and the variable domain of a light chain.
- variable domain interacts to determine the target binding site on the surface of the VH-VL dimer, as in the case of an intact antibody.
- the six CDRs together confer target binding specificity to the intact antibody.
- a single variable domain or half of an Fv that includes only three target-specific CDRs can still have the ability to recognize and bind to a target.
- monoclonal antibody refers to a colony of identical antibodies, representing that each individual antibody molecule in the monoclonal antibody colony is identical to other antibody molecules. This characteristic is contrary to the characteristic of the polyclonal colony of antibodies, which comprises antibodies with a variety of different sequences.
- Monoclonal antibodies can be prepared by many well-known methods. For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by merging with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal colony of host cells in vitro by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding antibodies.
- a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies produced synthetically with the same domains.
- chimeric antibody refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from mouse antibodies and the constant region sequences are derived from human antibodies.
- Humanized antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab') 2 , or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
- humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementary determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
- CDRs complementary determining regions
- PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
- the mutations in the CDR are generally no more than one or two. Therefore, the humanized antibodies described in the present disclosure also encompass antibodies comprising 1 or 2 amino acid mutations in the CDR.
- CDR refers to complementarity-determining region
- each heavy chain and light chain of an antibody molecule is known to have three CDRs.
- CDR is also called a hypervariable region and is present in the variable region of each heavy chain and light chain of an antibody, with very high variability sites in the primary structure of the CDR.
- the CDR of the heavy chain is represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the heavy chain
- CDR of the light chain is represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.
- epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
- Epitopic determinants usually comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
- telomere binding As used herein, "specific binding” or “immunospecifically binding” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen.
- the antigen may be an isolated antigen or present in a tumor cell.
- an antibody that immunospecifically binds (or specifically binds) to an antigen has an affinity constant Ka of about or 1 ⁇ 10 7 M -1 or 1 ⁇ 10 8 M -1 or greater (or a dissociation constant of 1 ⁇ 10 -7 M or 1 ⁇ 10 -8 M or less).
- the affinity constant (Kd) can be determined by standard kinetic methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available.
- nucleic acid As used herein, the terms “polynucleotide”, “nucleic acid” or “nucleic acid molecule” are used interchangeably and refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by a phosphodiester bond.
- nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
- expression refers to the process of producing a polypeptide by transcription and translation of a polynucleotide.
- the expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
- host cell is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid.
- the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
- vector is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
- Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation.
- Vectors also include those containing nucleic acids encoding polypeptides.
- Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression/display of polypeptides encoded by nucleic acids.
- Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
- vectors also include “viral vectors” or “viral vectors.”
- Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) the exogenous genes into cells.
- expression vector includes vectors capable of expressing DNA that is operably linked to regulatory sequences such as promoter regions that are capable of affecting the expression of such DNA fragments.
- the expression vector may be a recombinant DNA or RNA construct, such as a plasmid, a phage, a recombinant virus or other vector, which, when introduced into a suitable host cell, results in the expression of the cloned DNA.
- Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and/or prokaryotic cells and expression vectors that remain free or that are integrated into the host cell genome.
- diagnosis refers to an assessment of the likelihood that an individual has a disease or condition or is at risk of developing a disease or condition.
- assessment while preferably correct for 100% of the subjects to be diagnosed, is generally not the case.
- the term requires that a statistically significant portion of the subjects can be identified as having a disease or having a predisposition thereto.
- treating an individual suffering from a disease or condition means that the symptoms of the individual are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and/or cure. As used herein, “prevention” refers to preventing potential diseases and/or preventing symptoms from worsening or disease development. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided and any composition provided herein.
- therapeutic effect refers to the effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.
- FIG1 shows the binding activity of the hybridoma clone supernatant of the present invention to hCLDN18.2 and hCLDN18.1, wherein:
- FIG2 shows the comparison of the binding activities of the D11 chimeric antibody 425 of the present invention and reported antibodies (224, 224-1 and 435) to CLDN18.2.
- FIG3 shows the binding activity of the D11 humanized antibody 432 of the present invention to HEK293/203B3 cells.
- FIG. 4 shows the positions of amino acid mutations during the humanization of D11 of the present invention.
- FIG. 5 shows the binding activity of different mutant clones of the present invention to CLDN18.2.
- FIG6 shows the alignment of the first extracellular loop amino acids of hCLDN18.2 and hCLDN18.1, as well as mutation positions P1 to P8.
- FIG. 7 shows a comparison of the expression intensity of human CLDN18.2 in different engineered cell lines.
- FIG8 shows a comparison of the binding ability of the 432-M147-full antibody of the present invention to different human CLDN18.2 overexpressing cell lines, wherein:
- FIG. 9 shows the cross-binding activity of the 432-M147 whole antibody of the present invention with human CLDN18.1.
- FIG. 10 shows the cross-binding activity of the 432-M147 whole antibody of the present invention with mouse CLDN18.2.
- FIG. 11 shows the endocytic activity of the 432-M147 whole antibody of the present invention.
- FIG. 12 shows the CDC activity of the 432-M147 whole antibody of the present invention.
- FIG13 shows the activity of the 432-M147 full antibody of the present invention in ADCC reporter cell lines, wherein:
- MC38-hCLDN18.2-A11 is the target cell
- FIG. 14 shows the ADCC activity of 432-M147 of the present invention mediated by primary NK cells.
- FIG. 15 shows the ADCP activity mediated by 432-M147 of the present invention based on primary BMDM cells.
- mice 6-8 week old Balb/c mice were immunized with HEK293 (B3) cells stably expressing human CLDN18.2 (SEQ ID NO 2). Specifically, on the first day, 2x10 ⁇ 6 HEK293 cells were intraperitoneally injected into the mice for pre-immunization, and then on the second and third days, 200 mg/kg of cyclophosphamide was intraperitoneally injected into the mice to suppress the anti-HEK293 immunity of the mice. On days 14, 15, 29, 30, 64, and 65, mice were immunized with 2x10 ⁇ 6 B3 cells by intraperitoneal injection. Finally, on day 88, mice were immunized with 3x10 ⁇ 6 B3 cells by intraperitoneal injection. On day 91, spleens of mice were taken for cell fusion experiments.
- hybridoma technology and cell-based ELISA assays were used to obtain hybridomas that bind to B3 but not to HEK293 parental cells.
- the binding ability of hybridoma cell culture supernatants to B3 cells, HEK293 cells expressing human CLDN-18.1 (SEQ ID NO: 1) (HEK293/202) and HEK293 parental cells was compared by single cell cloning and flow cytometry, and CLDN18.2 monoclonal hybridomas, such as hybridomas 6H5D5 and D11, were selected, and the results are shown in Figures 1A and 1B, respectively.
- cDNA as a template, PCR was used to amplify the antibody VL and VH regions. After the PCR products were separated by 1% agarose gel electrophoresis, the DNA fragments were purified and recovered and cloned into the TA vector. The fragments were then sequenced to obtain the D11VL (SEQ ID NO: 3) and D11VH (SEQ ID NO: 4) sequences.
- the three light chain CDR sequences of D11 that were finally determined are: LCDR1 (SEQ ID NO: 5), LCDR2: WAS (SEQ ID NO: 6), and LCDR3 (SEQ ID NO: 7); and the three heavy chain CDR sequences are: HCDR1 (SEQ ID NO: 8), HCDR2 (SEQ ID NO: 9), and HCDR3 (SEQ ID NO: 10).
- Example 2 Construction of anti-CLDN18.2 antibodies and their expression in eukaryotic cells
- VL (SEQ ID NO: 3) and VH (SEQ ID NO: 4) of D11 were constructed into a vector containing hIgG1Fc by homologous recombination, thus obtaining chimeric antibody 425 expressing anti-CLDN18.2.
- chimeric antibodies ch-163E12 (WO2014/146778A1), ch-175D10 (WO2014/146778A1) and ch-CL-1 (WO2020/025792A1) plasmids 224 (163E12), 224-1 (175D10, Zolbetuximab (IMAB362)), and 435 (CL-1) were obtained by homologous recombination.
- plasmids were then transduced into 293T cells using the transfection reagent PEI, and the supernatant was collected after 64 hours, and the above antibodies were purified using Protein A.
- the corresponding fusion antibodies 224, 224-1, and 435 were obtained, and the relative affinity of these fusion antibodies was detected using cell B3 expressing human CLDN18.2.
- the EC50 of 425 was 0.29 nM, while the EC50 values of the known chimeric antibodies 224, 224-1, and 435 were 47.52 nM, 1.99 nM, and 1.78 nM, respectively, indicating that the D11 chimeric antibody has a strong affinity for human CLDN18.2.
- the inventors used D11VL and D11VH known antibody databases for search and sequence alignment (http://www.imgt.org DomainGapAlign), respectively, and selected GKV4-1*01 in the IMGT database as the template for the humanized antibody light chain, and IGHV4-59*01 as the antibody template for the humanized antibody heavy chain.
- the light chain and heavy chain CDR regions of 425 replaced the CDR regions of the antibody template, and then according to the homologous recombination instructions, they were homologously recombined into the plasmid 210 HindIII/XhoI site to obtain plasmid 432 (SEQ ID NO:15 and SEQ ID NO:17).
- the relative affinity EC50 of humanized antibody 432 for binding to HEK293/203B3 cells was 11.95nM.
- 432 has a lower affinity for CLDN18.2 (SEQ ID NO: 14 and SEQ ID NO: 16).
- the humanization process often leads to a decrease in the affinity of humanized antibodies.
- the inventors used 432 as a template for site-directed mutagenesis.
- 9 amino acids were identified in the 432 VL and VH regions that were inconsistent with the conserved amino acids at the corresponding positions of the parent or were modification hotspots ( Figure 4).
- the conserved amino acids at these positions may play an important role in the structure and function of VL and VH.
- the hydrophobic amino acid at position 2 and the positively charged K at position 7 in the parent sequence are essential for stabilizing the antibody loop structure.
- S at positions 4 and 8 and F at position 9 may participate in the interaction between VL and VH.
- a at position 3 and W at position 5 may participate in hydrogen bonding. Mutations in these conserved amino acids will lead to changes in affinity.
- D followed by a G at position 6 creates a potential D isomerization site DG.
- M at position 1 has the potential to be easily oxidized. Mutations of these amino acids will improve protein production, stability and drugability, and reduce the potential risks of antibody fragmentation and isomerization.
- a point mutation kit was used to perform site-directed mutagenesis on humanized antibody 432, and single-site mutants of humanized antibody 432 were obtained: 432-M1, 432-M2b, 432-M3, 432-M4, 432-M5, 432-M6, 432-M7, 432-M8b, 432-M8c, 432-M9, and multi-site mutants 432-M13, 432-M14, 432-M34, 432-M134, 432-M147, 432-M59, 432-M68c.
- an antibody depends largely on the epitope to which it binds to the target antigen.
- the epitope is a part of the antigen that binds to the variable region of the antibody; antibodies binding to different epitopes may produce different functions, which may manifest as inhibitory activity or activation of a certain signal, etc., so antigen epitope localization is particularly important.
- CLDN18.1 and CLDN18.2 are two different splice mutants, and their expression in different cells is significantly different. In tumor cells, CLDN18.2 protein is highly expressed, while CLDN18.1 is not expressed.
- the present invention uses a site-directed mutagenesis method to modify the quality of the protein.
- the 8 amino acids of CLDN18.2 protein were mutated into the amino acids at the corresponding positions of CLDN-18.1, and 8 plasmids P1 (Q29M), P2 (N37D), P3 (A42S), P4 (N45Q), P5 (Q47E), P6 (E56Q), P7 (G65P) and P8 (L69I) were obtained. These plasmids were transferred to HEK293T. Thereafter, HEK-293T cells expressing CLDN-18.1 and wild-type or mutant CLDN-18.2 were used for fusion antibody binding assays.
- 224-1, 435, 425, 432 and 432-mutants all specifically bind to HEK-293T (203) expressing CLDN18.2, whereas they do not bind to HEK-293T (202) expressing CLDN-18.1 or to the parental HEK-293T.
- Amino acid mutations at P1, P2, P5, P7 and P8 do not affect their binding, indicating that amino acids at these CLDN-18.2 positions are not involved in antibody binding.
- amino acid mutations at P4 and P6 completely abolished binding to all antibodies tested. Therefore, amino acids at CLDN18.2 P4 (N45) and P6 (E56) are key epitope amino acids for specific interaction of anti-CLDN18.2 antibodies.
- amino acids at P4 and P6 are also key sites for binding of 224-1 (IMAB362).
- the binding of another known fusion antibody 435 (CL-1) and the monoclonal antibody 425 of the present invention is not affected by the mutation of the CLDN18.2 P3 position.
- D11 and its humanized antibody and its mutants showed different sensitivities to CLDN18.2 P3: 432, 432-M8c, and 432-M9 had decreased binding; 432-M8b completely lost binding.
- the P3 (A42) mutation site has different effects on different CLDN18.2 monoclonal antibodies, indicating that this position may play a role in stabilizing the antibody structure.
- the signal peptide containing secretion signal (SP2, SEQ ID NO: 30) was introduced into the N-terminus of the variable region sequence of VL and VH of 432-M147, and SP2-LC was transformed into The light chain 432-M147-LC (SEQ ID NO: 24 and SEQ ID NO: 25) of the 432-M147 antibody was constructed on the plasmid pCDNA3.4 containing the light chain constant region fragment, and the heavy chain 432-M147-HC (SEQ ID NO: 26 and SEQ ID NO: 27) of the 432-M147 antibody was obtained by homologous recombination. Then, these plasmids were transferred into 293T cells through the transfection reagent PEI, and the supernatant was collected after 5 days, and the above IgG1 antibody was purified with Protein A.
- hCLDN18.2 In order to study the activity of 432-M147 pan-antibody, the inventors compared 5 different genetically engineered cell lines including the engineered cell lines CT26-hCLDN18.2 (constructed by our company), MC38-hCLDN18.2-C4 (constructed by our company), MC38-hCLDN18.2-A11 (constructed by our company), KATO III-hCLDN18.2 (cell number KC-1453, purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.) and 293T-hCLDN18.2 cells (cell number - KC-0986) that overexpress human CLDN18.2 (hereinafter referred to as hCLDN18.2).
- the present inventors used 6.67nM-432-M147 antibody to incubate 8x 10 ⁇ 4 of the above-mentioned different cells, added 1:100 diluted PE-labeled secondary antibody (PE anti-human IgG Fc antibody, Biolegend) staining solution, added 100 ⁇ L secondary antibody staining solution to the experimental group and the secondary antibody control group, and added an equal volume of flow buffer to the blank control well; after mixing, incubate at 4°C in the dark for 1 hour; wash the cells twice with flow buffer, 200 ⁇ L each time, centrifuge at 1500r/min for 4 minutes.
- PE-labeled secondary antibody PE anti-human IgG Fc antibody, Biolegend
- the inventors used these cell lines carrying different membrane surface CLDN18.2 molecular densities to conduct comprehensive binding activity tests on 432-M147 and the reference antibody Zolbetuximab.
- the experimental steps were as described above, except that the concentration of the 432-M147 antibody started from 667 nM and was diluted 3.16 times to 8 concentrations, and the negative controls were hIgG1 and blank secondary antibody controls.
- the PE signal was detected by NovoCyte Quanteon flow cytometer (Agilent), and the data were analyzed by GraphPad Prism 7.0 software.
- the nonlinear S-curve four-parameter fitting regression was used to fit the data to obtain the dose-effect curve:
- Y Bottom + (Top-Bottom) / (1 + 10 ⁇ ((LogEC 50 -X)*HillSlope)), where Bottom represents the lower limit of the fluorescence signal, representing the lower asymptote of the S curve, and Top represents the upper limit of the fluorescence signal, representing the upper asymptote of the S curve.
- EC 50 is the concentration value at which the growth rate of the fluorescence signal begins to change, that is, the half-effective reaction concentration (EC 50 ), and the EC 50 value is calculated from it.
- HillSlope is the absorbance increase rate parameter, which is equivalent to the slope of the curve, and the IC50 value is calculated from it.
- Table 4 Summary of the binding activity of 432-M147 in cell lines with different human CLDN18.2 antigen strengths
- 432-M147 showed a clear affinity advantage. Although no effective EC 50 was obtained, the inventors found that the binding activity of 432-M147 showed a concentration gradient-dependent increase, while the reference antibody Zolbetuximab had no binding activity at all under low antigen density.
- the binding activity EC50 of 432-M147 and Zolbetuximab were 4.7 nM and 17.8 nM, respectively, with a 3.8-fold difference in affinity; the maximum binding fluorescence intensity Emax of 432-M147 was 6.7 times higher than that of the reference antibody.
- the binding activity EC50 of 432-M147 and Zolbetuximab were 8.2nM and 57.8nM, respectively, with an affinity increased by 7 times, and the maximum binding fluorescence intensity Emax of 432-M147 was 6.5 times higher than that of the reference antibody; in another high antigen density cell KATOIII-hCLDN18.2, as shown in Figure 8E, the binding activity EC50 of 432-M147 and Zolbetuximab were 5.7nM and 9.7nM, respectively, with an affinity difference of 1.7 times, and the maximum binding fluorescence intensity Emax of 432-M147 was 2.6 times higher than that of the reference antibody.
- the 432-M147 antibody of the present invention has a significantly improved affinity compared with the reference antibody Zolbetuximab on cell lines with different antigen expression intensities, especially in the case of low antigen abundance. Due to the heterogeneity of CLDN18.2 tumor antigen in tumor expression, the high affinity binding of 432-M147 at low CLDN18.2 antigen abundance indicates that this antibody can be used for the treatment of patients with low expression of CLDN18.2 tumor antigen compared with the reference antibody, which will provide an important theoretical basis for subsequent applications.
- CLDN18.1 and CLDN18.2 belong to the CLDN18 family, with a sequence homology of about 92% (240/261), and only 21 amino acids differ in the first transmembrane region of the N-terminus (Sahin et al., 2008).
- the present inventors tested the binding activity of the 432-M147 antibody to the human CLDN18.1 antigen.
- the experimental method was similar to that of Example 5, except that the cells used were engineered cell lines 293T-hCLDN18.1 cells (purchased from Kangyuan Bochuang, cell number-KC-0990) that highly express human CLDN18.1 (hereinafter referred to as hCLDN18.1) protein.
- Table 5 Summary of the binding activity of 432-M147 antibody to mouse CLDN18.2
- Receptor-mediated endocytosis is an important pathway for many membrane proteins to mediate the entry of antibodies and antibody-drug conjugates into cells, so the present inventors evaluated the endocytic activity of 432-M147.
- MC-38-hCLDN18.2-A11 cells in the logarithmic growth phase were collected, centrifuged at 1000 rpm/min for 5 minutes, and the supernatant was discarded.
- the cells were washed once with PBS, resuspended in flow cytometry buffer and counted, and the density was adjusted to 5 ⁇ 10 ⁇ 6 cells/mL and added to the flow tube.
- the test antibody was added with a working final concentration of 66.7 nM, and incubated at 4°C in the dark for 1 hour.
- the cells were washed twice with pre-cooled flow cytometry buffer, and the cells were resuspended with 100 ⁇ L of washing solution and 2 ⁇ L of PE-labeled secondary antibody (PE anti-human IgG Fc antibody) was added.
- the cells were incubated at 4°C in the dark for 40 minutes, washed twice with pre-cooled flow cytometry buffer, and resuspended in 200 ⁇ L complete culture medium (DMEM+10% FBS) with 100 ⁇ L of washing solution, and cultured in a 37°C incubator for 2 hours, 1 hour, 0.5 hours, and 0 hours, respectively.
- DMEM+10% FBS complete culture medium
- Dissociation Wash cells once with flow cytometry buffer, resuspend cells with 200ul of dissociation buffer, and incubate at room temperature for 7min.
- Neutralization Add neutralization buffer and centrifuge, and finally resuspend cells with 100 ⁇ L of flow cytometry buffer. PE signal was detected by NovoCyte Quanteon flow cytometer (Agilent). The experimental results are shown in Figure 11 and Table 6 (the fold difference was calculated by the internalization rate of 432-M147 at each time point/the internalization rate of the reference antibody Zolbetuximab Come).
- Complement-dependent cytotoxicity is the mechanism of action of many antibodies causing tumor killing.
- the Fab end of the antibody binds to the antigen
- the Fc end of the antibody binds to the complement C1q in the serum, activating the complement system to form a membrane attack complex to exert a lytic effect on the target cell. Therefore, the inventors further compared the CDC activity of 432-M147 and the reference antibody Zolbetuximab.
- KATOIII-hCLDN18.2 purchased from Kangyuan Bochuang, cell number-KC-1453 cells were centrifuged and counted, and 1*10 ⁇ 5 cells per well were inoculated into a U-shaped bottom 96-well plate.
- Antibody-dependent cell-mediated cytotoxicity is the main mechanism of action for many antibody drugs to kill tumor cells.
- Fc ⁇ RIIIA CD16a
- Fc ⁇ RIIIA CD16a
- NK cells NK cells on tumor cells.
- Jurkat-NFAT-Luc2-CD16a-V15 constructed by Kangyuan Bochuang
- KATO III CLDN18.2 cells purchased from Kangyuan Bochuang, cell number KC-1453 overexpressing human CLDN18.2 at a ratio of 1:1.
- the extracellular region of this effector cell overexpressed high-affinity Fc ⁇ RIIIA CD16a-V158 and was co-incubated with target cells and different concentrations of test antibodies (starting at 133.4nM, 5-fold dilution, 9 concentration points).
- the antibody Fc end binds to the high-affinity CD16a-V158 in the extracellular region to activate the NFAT-luc2 luciferase reporter system.
- the ADCC activity of the antibody can be detected by detecting the content of luciferase using a Plus multifunctional microplate reader (BMG LABTECH).
- the data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was obtained by fitting the data using nonlinear S-curve regression, and the EC 50 value was calculated from this.
- the experimental results are shown in Figures 13A-13B and Table 8.
- Table 8 Summary of ADCC reporter cell line activity of 432-M147 antibody
- the fluorescence signal of the Jurkat-NFAT-Luc2-CD16a-V158 reporter cell line showed that in the high-density cell line MC38-hCLDN18.2-A11, the ADCC activity of 432-M147 was significantly better than that of the reference antibody Zolbetuximab, with EC50 of 0.08nM and 1.8nM, respectively.
- the ADCC activity of 432-M147 was also significantly better than that of the reference antibody Zolbetuximab, with EC50 of 0.03nM and 2nM, respectively.
- the ADCC activity of 432-M147 was 22.5 times and 66.7 times higher than that of the reference antibody Zolbetuximab, respectively, indicating that the ADCC activity of 432-M147 was significantly improved.
- 432-M147 can inhibit the killing of leukemia cells through the antibody-mediated complement killing pathway and the The antibody-mediated cytotoxicity kills CLDN18.2 antigen-positive tumor cells, showing its clinical application value is superior to the reference antibody.
- ADCC is caused by the Fab end of the antibody binding to the antigen epitope of the tumor cell, and its Fc end binding to the FC ⁇ R on the surface of the natural killer cell (NK cell).
- the NK cell is activated to release cytotoxic substances such as perforin and granzyme, which mediate the killing of target cells by NK cells and cause apoptosis of target cells.
- the inventors used primary NK cells derived from peripheral blood mononuclear cells PBMC as effector cells and used 293T-hCLDN18.2 as target cells to establish a co-culture system to simulate ADCC in vitro.
- the target cells 293T-hCLDN18.2 (purchased from Kangyuan Bochuang, cell number-KC-0986) were labeled with 1.6 ⁇ M CFSE (565082, BD), labeled for 10 minutes at room temperature in the dark, washed twice with 5 times the volume of pre-cooled serum-free medium, resuspended in ADCC culture medium and counted, and 5x 10 ⁇ 4 cells were added to each well of the 96-well U-shaped plate, and then the test antibody was added for gradient dilution, with a starting concentration of 333.5nM, gradient dilution of 3.16 times with complete medium, a total of 10 concentration points, centrifuged at 500 rpm for 30 seconds, and then 1.5x 10 ⁇ 5 PBMC cells/well were added, with an effector-target ratio of 30:1.
- the target cell effector cell antibody complex was incubated at 37°C for 4 hours. After the incubation, the cells were washed twice with flow cytometry buffer, 100 ⁇ L of PBS solution containing 1 ⁇ L 7-AAD (559925, BD) was added to each well for staining, incubated at room temperature in the dark for 10 minutes, and the activity of the target cells was detected using a NovoCyte Quanteon flow cytometer (Agilent). The data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was fitted using nonlinear S-curve regression, and the EC 50 value was calculated from this. The experimental results are shown in Figure 14 and Table 9.
- Table 9 Summary of primary NK cell-mediated ADCC activity of 432-M147 antibody
- the experimental results showed that in the primary NK cell-mediated ADCC killing experiment, similar to the results of the reported cell line, 432-M147 still showed strong ADCC activity with an EC 50 of 0.18nM, while the EC 50 of the reference antibody Zolbetuximab was 0.5nM.
- the primary NK cell-mediated ADCC activity of 432-M147 increased by 2.7 times, indicating that its tumor killing activity is superior to that of Zolbetuximab.
- the ADCC activity mediated by Jurkat-NFAT-Luc2-CD16a-V158 reporter cell line and primary NK cells showed that the ADCC activity of 432-M147 of the present invention was significantly higher than that of the reference antibody.
- the activity has been significantly improved.
- the ADCC activity of antibodies is an important tumor killing mechanism. High ADCC activity will provide an important theoretical basis for clinical applications.
- Antibody-dependent cell-mediated phagocytosis is another important mechanism for antibody drugs to kill tumor cells.
- the Fab of the antibody binds to the antigen, causing conformational changes, and promoting the binding of the Fc end of the antibody to Fc ⁇ RIIA (CD32a) on the surface of macrophages, and activating the downstream signal ITAM signal of Fc ⁇ RIIA, promoting the phagocytosis of tumor antigen-positive cells by macrophages.
- the inventors used macrophages derived from mouse bone marrow differentiation and co-cultured with 293T-hCLDN18.2 cells to evaluate the ADCP activity of the 432-M147 antibody.
- the induced BMDM was digested with 1mL accutase, centrifuged at 1200rpm for 5 minutes, and resuspended and counted with 1640+5% FBS for standby use.
- the target cells were labeled with CFSE at a final concentration of 1.6 ⁇ M/mL, resuspended and counted in 1640+5% FBS for staining at room temperature in the dark, and plated on a low-absorption 96-well U-bottom plate at 5X10 ⁇ 4 cell/well, 80 ⁇ L per well.
- the induced BMDM was plated on a low-absorption 96-well U-bottom plate according to the corresponding effector-target ratio, 80 ⁇ L per well. After mixing with a shot gun, the cells were incubated at 37 degrees for 4 hours.
- FUT8 knockout HEK293/FUT8 - cells 432-M147 the cells were purchased from Kangyuan Bochuang Biotechnology Co., Ltd. (KC-2300).
- the cell line was subcultured in Expi293 TM expression medium for production, with a subculture density of 0.2-0.3*E6/ml and a subculture cycle of 2-3 days. Dilute the cells to 2*E6/ml the day before transfection. On the day of transfection, the cell density should be around 6E6/ml and the viability should be greater than 95%. On the day of transfection, follow the instructions of the transfection kit for transfection.
- Detection wavelengths of 2-AB labeling are: excitation wavelength 330nm, emission wavelength 330nm.
- the method for detecting the ADCC activity of ICP is as described in Example 13, using 293T-hCLDN18.2 (cells purchased from Kangyuan Bochuang, cell number KC-0986) cells as target cells and co-cultured with primary NK cells derived from PBMC to detect ADCC activity mediated by the Fc end of the fusion protein.
- the 432-M147 antibody of the present invention has significantly improved affinity, endocytosis activity, CDC activity, ADCC activity and ADCP activity.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
Abstract
L'invention concerne un anticorps anti-CLDN18.2 ou un fragment de liaison à l'antigène de celui-ci, un polynucléotide codant pour l'anticorps anti-CLDN18.2 ou le fragment de liaison à l'antigène de celui-ci, un vecteur, une cellule hôte, et un procédé de préparation, ainsi qu'une composition pharmaceutique pour le diagnostic, la prévention et/ou le traitement d'une tumeur liée à CLDN18.2 et son utilisation.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211199798.8A CN117777288A (zh) | 2022-09-29 | 2022-09-29 | 抗密蛋白18.2的抗体 |
CN202211199798.8 | 2022-09-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024067612A1 true WO2024067612A1 (fr) | 2024-04-04 |
Family
ID=90385660
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2023/121643 WO2024067612A1 (fr) | 2022-09-29 | 2023-09-26 | Anticorps anti-claudine 18.2 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN117777288A (fr) |
WO (1) | WO2024067612A1 (fr) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019174617A1 (fr) * | 2018-03-14 | 2019-09-19 | Beijing Xuanyi Pharmasciences Co., Ltd. | Anticorps anti-claudine 18.2 |
CN111836644A (zh) * | 2018-03-08 | 2020-10-27 | 东莞凡恩世生物医药有限公司 | 抗密蛋白18.2抗体及其用途 |
WO2021047599A1 (fr) * | 2019-09-13 | 2021-03-18 | Beijing Xuanyi Pharmasciences Co., Ltd. | Anticorps humanisés anti-claudine 18.2 (cldn18.2) |
CN112912396A (zh) * | 2018-10-22 | 2021-06-04 | 上海吉倍生物技术有限公司 | 抗cldn18.2抗体及其用途 |
CN113166246A (zh) * | 2018-12-28 | 2021-07-23 | 四川科伦博泰生物医药股份有限公司 | 一种抗体及其用途 |
US20220041712A1 (en) * | 2019-02-01 | 2022-02-10 | Novarock Biotherapeutics, Ltd. | Anti-claudin 18 antibodies and methods of use thereof |
-
2022
- 2022-09-29 CN CN202211199798.8A patent/CN117777288A/zh active Pending
-
2023
- 2023-09-26 WO PCT/CN2023/121643 patent/WO2024067612A1/fr unknown
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111836644A (zh) * | 2018-03-08 | 2020-10-27 | 东莞凡恩世生物医药有限公司 | 抗密蛋白18.2抗体及其用途 |
WO2019174617A1 (fr) * | 2018-03-14 | 2019-09-19 | Beijing Xuanyi Pharmasciences Co., Ltd. | Anticorps anti-claudine 18.2 |
CN112912396A (zh) * | 2018-10-22 | 2021-06-04 | 上海吉倍生物技术有限公司 | 抗cldn18.2抗体及其用途 |
CN113166246A (zh) * | 2018-12-28 | 2021-07-23 | 四川科伦博泰生物医药股份有限公司 | 一种抗体及其用途 |
US20220041712A1 (en) * | 2019-02-01 | 2022-02-10 | Novarock Biotherapeutics, Ltd. | Anti-claudin 18 antibodies and methods of use thereof |
WO2021047599A1 (fr) * | 2019-09-13 | 2021-03-18 | Beijing Xuanyi Pharmasciences Co., Ltd. | Anticorps humanisés anti-claudine 18.2 (cldn18.2) |
Also Published As
Publication number | Publication date |
---|---|
CN117777288A (zh) | 2024-03-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230120270A1 (en) | New polypeptide complex | |
CN111171155B (zh) | 抗cd3和cd123双特异性抗体及其用途 | |
TWI830151B (zh) | 抗GPRC5DxBCMAxCD3三特異性抗體及其用途 | |
EP4289862A1 (fr) | Anticorps anti-b7-h3 humain et utilisation associée | |
WO2021169982A1 (fr) | Anticorps ciblant epcam, et procédé de préparation et application associés | |
WO2021143914A1 (fr) | Anticorps anti-ox40, son procédé de production et son application | |
EP4303234A1 (fr) | Anticorps dirigés contre nkp46 et application d'anticorps | |
WO2022100694A1 (fr) | Anticorps et son procédé de préparation | |
WO2024067612A1 (fr) | Anticorps anti-claudine 18.2 | |
TWI815136B (zh) | 一種雙特異性抗體及其用途 | |
WO2024067759A1 (fr) | Anticorps capable de se lier à cldn18.2 ou fragment de liaison à l'antigène de celui-ci et son utilisation | |
WO2024094017A1 (fr) | Anticorps bispécifique pour glypicane-3 et son utilisation | |
WO2023036215A1 (fr) | Molécule de liaison à un antigène bispécifique et son utilisation | |
WO2023016450A1 (fr) | Anticorps anti-tigit et son utilisation | |
WO2024027815A1 (fr) | Molécule de liaison à l'antigène se liant spécifiquement à gucy2c et cd3 et son utilisation pharmaceutique | |
US20230331862A1 (en) | Antibody specifically bound to glycosylated ceacam5 | |
WO2022247826A1 (fr) | Protéine de liaison spécifique ciblant pd-l1 et cd73 | |
WO2023208182A1 (fr) | Anticorps anti-ccr8 et son utilisation | |
TW202413437A (zh) | 特異性結合gucy2c和cd3的抗原結合分子及其醫藥用途 | |
TW202413414A (zh) | 抗ilt4抗體及其醫藥用途 | |
TW202405004A (zh) | 特異性結合dll3和cd3的抗原結合分子及其醫藥用途 | |
KR20240015616A (ko) | CLDN18.2에 대한 단클론 항체 및 이의 Fc 조작된 형태 | |
CN117229412A (zh) | 包含抗cldn18.2抗体的双特异性抗体、药物组合物及用途 | |
CN114426580A (zh) | 抗csf-1r抗体及其产品、方法和用途 |
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: 23870840 Country of ref document: EP Kind code of ref document: A1 |