WO2021020564A1 - Gd2結合性分子 - Google Patents
Gd2結合性分子 Download PDFInfo
- Publication number
- WO2021020564A1 WO2021020564A1 PCT/JP2020/029446 JP2020029446W WO2021020564A1 WO 2021020564 A1 WO2021020564 A1 WO 2021020564A1 JP 2020029446 W JP2020029446 W JP 2020029446W WO 2021020564 A1 WO2021020564 A1 WO 2021020564A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- seq
- amino acid
- acid sequence
- heavy chain
- Prior art date
Links
- 230000027455 binding Effects 0.000 title claims abstract description 69
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 71
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 22
- 201000011510 cancer Diseases 0.000 claims abstract description 14
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 220
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 89
- 101000597780 Mus musculus Tumor necrosis factor ligand superfamily member 18 Proteins 0.000 claims description 35
- 102100035283 Tumor necrosis factor ligand superfamily member 18 Human genes 0.000 claims description 35
- 108091033319 polynucleotide Proteins 0.000 claims description 32
- 239000002157 polynucleotide Substances 0.000 claims description 32
- 102000040430 polynucleotide Human genes 0.000 claims description 32
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 27
- 239000008194 pharmaceutical composition Substances 0.000 claims description 22
- 230000003834 intracellular effect Effects 0.000 claims description 21
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 20
- 150000002270 gangliosides Chemical class 0.000 claims description 18
- 210000004899 c-terminal region Anatomy 0.000 claims description 13
- 210000000822 natural killer cell Anatomy 0.000 claims description 6
- PFJKOHUKELZMLE-VEUXDRLPSA-N ganglioside GM3 Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@@H]([C@H](O)/C=C/CCCCCCCCCCCCC)NC(=O)CCCCCCCCCCCCC\C=C/CCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 PFJKOHUKELZMLE-VEUXDRLPSA-N 0.000 claims description 5
- CXQCLLQQYTUUKJ-ALWAHNIESA-N beta-D-GalpNAc-(1->4)-[alpha-Neup5Ac-(2->8)-alpha-Neup5Ac-(2->3)]-beta-D-Galp-(1->4)-beta-D-Glcp-(1<->1')-Cer(d18:1/18:0) Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@@H](CO)O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 CXQCLLQQYTUUKJ-ALWAHNIESA-N 0.000 claims description 4
- WPIHMWBQRSAMDE-YCZTVTEBSA-N beta-D-galactosyl-(1->4)-beta-D-galactosyl-N-(pentacosanoyl)sphingosine Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC(=O)N[C@@H](CO[C@@H]1O[C@H](CO)[C@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O)[C@H](O)\C=C\CCCCCCCCCCCCC WPIHMWBQRSAMDE-YCZTVTEBSA-N 0.000 claims description 4
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 claims description 3
- QPJBWNIQKHGLAU-IQZHVAEDSA-N ganglioside GM1 Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 QPJBWNIQKHGLAU-IQZHVAEDSA-N 0.000 claims description 3
- LEZNRPFLOGYEIO-QSEDPUOVSA-N ganglioside GT1b Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@@H](CO)O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@]4(O[C@H]([C@H](NC(C)=O)[C@@H](O)C4)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O3)O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 LEZNRPFLOGYEIO-QSEDPUOVSA-N 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 46
- 239000012636 effector Substances 0.000 description 33
- 230000014509 gene expression Effects 0.000 description 32
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 25
- 239000002585 base Substances 0.000 description 24
- 230000001472 cytotoxic effect Effects 0.000 description 21
- 239000013613 expression plasmid Substances 0.000 description 20
- 238000012258 culturing Methods 0.000 description 19
- 238000000034 method Methods 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 15
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 14
- 125000000539 amino acid group Chemical group 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 12
- -1 α-naphthyl Chemical group 0.000 description 12
- 231100000433 cytotoxic Toxicity 0.000 description 11
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 10
- 239000000427 antigen Substances 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 238000006467 substitution reaction Methods 0.000 description 10
- 101001023379 Homo sapiens Lysosome-associated membrane glycoprotein 1 Proteins 0.000 description 9
- 101150106931 IFNG gene Proteins 0.000 description 9
- 102100035133 Lysosome-associated membrane glycoprotein 1 Human genes 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 230000003013 cytotoxicity Effects 0.000 description 8
- 231100000135 cytotoxicity Toxicity 0.000 description 8
- 210000002325 somatostatin-secreting cell Anatomy 0.000 description 8
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- 108091008874 T cell receptors Proteins 0.000 description 7
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 7
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 125000005647 linker group Chemical group 0.000 description 7
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 7
- 231100001083 no cytotoxicity Toxicity 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 241001430294 unidentified retrovirus Species 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 5
- 108700005078 Synthetic Genes Proteins 0.000 description 5
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000009878 intermolecular interaction Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 201000001441 melanoma Diseases 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 125000002252 acyl group Chemical group 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 201000005202 lung cancer Diseases 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000007911 parenteral administration Methods 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 125000006850 spacer group Chemical group 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229930186217 Glycolipid Natural products 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010029260 Neuroblastoma Diseases 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 206010041067 Small cell lung cancer Diseases 0.000 description 3
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 3
- 108700012920 TNF Proteins 0.000 description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000021164 cell adhesion Effects 0.000 description 3
- 230000005859 cell recognition Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 238000010253 intravenous injection Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 201000008968 osteosarcoma Diseases 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 208000000587 small cell lung carcinoma Diseases 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 108010022379 (N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase Proteins 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 102100031505 Beta-1,4 N-acetylgalactosaminyltransferase 1 Human genes 0.000 description 2
- 101710098803 Beta-1,4 N-acetylgalactosaminyltransferase 1 Proteins 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 208000005243 Chondrosarcoma Diseases 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 101001019455 Homo sapiens ICOS ligand Proteins 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 2
- 102100034980 ICOS ligand Human genes 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 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 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 241000249107 Teschovirus A Species 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 208000024770 Thyroid neoplasm Diseases 0.000 description 2
- 206010062129 Tongue neoplasm Diseases 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 208000002495 Uterine Neoplasms Diseases 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000008043 acidic salts Chemical class 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000007942 carboxylates Chemical class 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000012212 insulator Substances 0.000 description 2
- 238000010212 intracellular staining Methods 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 235000014705 isoleucine Nutrition 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- 201000002510 thyroid cancer Diseases 0.000 description 2
- 201000006134 tongue cancer Diseases 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- 206010046766 uterine cancer Diseases 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 229960004295 valine Drugs 0.000 description 2
- 235000014393 valine Nutrition 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 1
- 125000006552 (C3-C8) cycloalkyl group Chemical group 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 125000000094 2-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- 108010082808 4-1BB Ligand Proteins 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 239000004475 Arginine 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
- 229940122361 Bisphosphonate Drugs 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 241000214054 Equine rhinitis A virus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-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
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101001042104 Homo sapiens Inducible T-cell costimulator Proteins 0.000 description 1
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical group C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 1
- 102100021317 Inducible T-cell costimulator Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 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
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 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
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-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
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-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
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 108010032605 Nerve Growth Factor Receptors Proteins 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241000609499 Palicourea Species 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241001420369 Thosea Species 0.000 description 1
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 description 1
- 102100033725 Tumor necrosis factor receptor superfamily member 16 Human genes 0.000 description 1
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000016571 aggressive behavior Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 239000003963 antioxidant 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
- 239000003899 bactericide agent Substances 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 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
- 239000011230 binding agent Substances 0.000 description 1
- 150000004663 bisphosphonates Chemical class 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 101150058049 car gene Proteins 0.000 description 1
- 125000001589 carboacyl group Chemical group 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000001511 cyclopentyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000003221 ear drop Substances 0.000 description 1
- 229940047652 ear drops Drugs 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000003885 eye ointment Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 150000002306 glutamic acid derivatives Chemical class 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000010954 inorganic particle Substances 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 235000005772 leucine Nutrition 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-M methanesulfonate group Chemical class CS(=O)(=O)[O-] AFVFQIVMOAPDHO-UHFFFAOYSA-M 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 108010056030 retronectin Proteins 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 125000002813 thiocarbonyl group Chemical group *C(*)=S 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 108010065816 zeta chain antigen T cell receptor Proteins 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
- C07K16/3076—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 against structure-related tumour-associated moieties
- C07K16/3084—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 against structure-related tumour-associated moieties against tumour-associated gangliosides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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 A61K40/00
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
-
- A61K39/4611—
-
- A61K39/4631—
-
- A61K39/464471—
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
-
- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
-
- 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
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- 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
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- 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
- C12N2510/00—Genetically modified 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/10041—Use of virus, viral particle or viral elements as a vector
- C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to GD2-binding molecules and the like.
- Ganglioside is a type of glycolipid and is composed of a sugar chain part and a lipid (ceramide: fatty acid + long chain base). Gangliosides are synthesized by a series of enzymatic reactions and metabolized to the final product.
- GD2 is synthesized from GD3 by GM2 / GD2 synthase, and further synthesized into GD1b by GM1 / GD1b / GA1 synthase.
- GD2 Since the expression of GD2 synthase is high and the expression of GD1b synthase is low in cancer cells, GD2 is highly expressed on the cell surface. It is known that GD2 expressed on cells coexists with adhesion molecules such as Integrin and is involved in cell adhesion and signal transduction, and is involved in cancer growth and metastasis.
- GD2 is known to be highly expressed in melanoma, neuroblastoma, glioblastoma, lung cancer, osteosarcoma and leukemia, and is expressed in nerve cells and glial cells in normal tissues, but these are normal. Low expression in tissues.
- Patent Document 1 Since GD2 is considered to be a good molecular target, an antibody that recognizes it has been isolated, and antibody treatment and CAR treatment have been performed (Patent Document 1), but the effect is limited and sufficient so far. The therapeutic effect has not improved. For example, as shown in Fig. 3 of Patent Document 1, even for P1143, which has a weak in vitro cytotoxic effect and the strongest effect, E: T ratio 5: 1 is about 20% lysis. Others are only a few percent. In addition, the same document describes a treatment experiment with 1x10 ⁇ 7 effector infusion after melanoma was administered IV to produce lung cancer (Fig. 6), but 20% died on day 100 and was completely cured. Not reached.
- An object of the present invention is to provide a cancer treatment or prevention technique targeting GD2 as a molecule.
- the present inventor shows heavy chain CDR1 containing the amino acid sequence shown by SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence shown by SEQ ID NO: 2, and SEQ ID NO: 3.
- Heavy chain variable region containing heavy chain CDR3 containing the amino acid sequence, and / or light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 9, light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 10, and SEQ ID NO: 11 It has been found that a GD2-binding molecule containing a light chain variable region containing a light chain CDR3 containing the amino acid sequence represented by the above can solve the above problems. As a result of further research based on this finding, the present invention has been completed. That is, the present invention includes the following aspects.
- a light chain variable region containing a light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 9 a light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 10
- GD2-binding molecule including.
- Item 2 The GD2-binding molecule according to Item 1, which comprises the heavy chain variable region and the light chain variable region.
- Item 3 The GD2 binding property according to Item 1 or 2, wherein the binding property to ganglioside GD1a, ganglioside GD1b, ganglioside GD3, ganglioside GM1, ganglioside GM3, ganglioside GT1b, and lactosylceramide is 1/2 or less of the binding property to ganglioside GD2. molecule.
- Item 4. The GD2-binding molecule according to any one of Items 1 to 3, which is a chimeric antigen receptor.
- Item 5 The GD2-binding molecule according to Item 4, which comprises the heavy chain variable region and the scFv domain containing the light chain variable region, the transmembrane domain, and the core region containing the intracellular domain of TCR.
- Item 6 The GD2-binding molecule according to Item 5, wherein the core region further contains an intracellular domain of a co-stimulator.
- Item 7. The GD2-binding molecule according to Item 5 or 6, which contains a GITRL domain via a self-cleaving peptide domain on the C-terminal side of the core region.
- Item 8 The GD2-binding molecule according to any one of Items 1 to 3, which is an antibody.
- Item 9 A polynucleotide encoding the GD2-binding molecule according to any one of Items 1 to 8.
- Item 10 A cell containing the polynucleotide according to item 9.
- Item 11 A chimeric antigen receptor T cell or a chimeric antigen receptor NK cell containing a polynucleotide encoding the GD2-binding molecule according to any one of Items 4 to 7.
- Item 3. A pharmaceutical composition containing the chimeric antigen receptor T cell or chimeric antigen receptor NK cell according to Item 11 or the GD2-binding molecule according to Item 8.
- Item 13 The pharmaceutical composition according to Item 12, which is used for treating or preventing cancer.
- cancer treatment or prevention technique targeting GD2 as a molecule it is possible to provide a cancer treatment or prevention technique targeting GD2 as a molecule.
- cancer can be treated or prevented by using an antibody that targets GD2 as a molecule, a chimeric antigen receptor that targets GD2 as a molecule, and the like.
- Lanes 1 and 2 in the photo on the right are lanes using ganglioside extract 3 microlitter, 1 microlitter of SK-MEL-23 (Carney2) cells (2 ml of glycolipid extracted from 10 g pellets C: (Dissolved in M (1: 1)), lanes 3 and 4 are lanes using AS cell extract 1 microliter, 3 microliter (0.5 ml of glycolipid extracted from 1 g pellet was C: M ( Dissolve in 1: 1)).
- the results of flow cytometry of Test Example 4 are shown.
- the vertical axis shows the number of counted cells, and the horizontal axis shows the fluorescence intensity of the cells. Black peaks indicate samples treated with 220-51 antibody and gray (red) peaks indicate samples not treated with 220-51 antibody.
- the cells used are shown above the histogram.
- AS, IMR32, Kohl-3 (SK-MEL-31), YTN17 are GD2-positive cells, and CEM, MOLT4 are GD2-negative cells.
- the RT-CES analysis results of Test Example 5 are shown.
- the vertical axis shows the Cell index (calculated from the electrical resistance), and the horizontal axis shows the elapsed time from the start of cell adhesion measurement.
- a schematic diagram of the structure of four types of CARs (28zCAR, zGCAR, 28zGITRLCAR, zGGITRLCAR) is shown.
- the results of Test Example 8 are shown (expression in ⁇ / ⁇ cells in the case of introduction of 28z CAR or 28z GITRL CAR expression plasmid).
- the expression efficiency of anti-kappa CAR is shown in%.
- the expression intensity of the CAR-expressing cell fraction is indicated by MFI.
- the results of Test Example 8 are shown (expression in alpha / beta cells in the case of introduction of zGCAR or zGGITRLCAR expression plasmid).
- the expression efficiency of anti-kappa CAR is shown in%.
- the expression intensity of the CAR-expressing cell fraction is indicated by MFI.
- the results of Test Example 8 are shown (expression in alpha / beta cells when CAR expression plasmid is not introduced).
- the expression efficiency of anti-kappa CAR is shown in%.
- the expression intensity of the CAR-expressing cell fraction is indicated by MFI.
- Test Example 8 The results of Test Example 8 are shown (expression in alpha / beta cells when 28z GITRL CAR expression plasmid is introduced). The percentage of GITRL-expressing cells is shown in%. The results of Test Example 8 are shown (expression in alpha / beta cells in the case of introduction of zG GITRL CAR expression plasmid). The percentage of GITRL-expressing cells is shown in%. The results of Test Example 8 are shown (in the case of CAR expression plasmid introduction, expression in alpha / beta cells). The percentage of GITRL-expressing cells is shown in%.
- Test Example 8 The results of Test Example 8 are shown (expression in gamma / delta cells when 28z CAR or 28z GITRL CAR expression plasmid is introduced and when CAR expression plasmid is not introduced). The percentage of kappa-positive and Vd2-positive fractions is shown. The results of Test Example 8 are shown (expression in gamma / delta cells when 28z GITRL CAR expression plasmid is introduced and when CAR expression plasmid is not introduced). The percentage of GITRL-expressing cells is shown in%.
- Gamma / delta cells, target cell recognition AS cells and CAR T cells were co-cultured for 4 hours, and then the intracellular expression IFNg and CD107a were measured with a flow cytometer (Test Example 9). Shown. The percentage of cells expressing IFNg, CD107a is shown in%. After co-culturing gamma / delta cells, target cell recognition AS cells and CAR T cells (28z GITRL CAR expression plasmid introduction) for 4 hours, intracellular expression IFNg, CD107a was measured with a flow cytometer (Test Example 9). Is shown. The percentage of cells expressing IFNg, CD107a is shown in%.
- the vertical axis shows the cytotoxic activity (%) measured by xCelligence, and the horizontal axis shows the elapsed time from the addition of effector cells.
- the results of the non-RI cytotoxicity test in Test Example 10 are shown.
- the vertical axis shows the percentage of injured cells calculated based on the amount of luminescence.
- the ratio in the legend indicates the ratio of CAR-T cells to AS cells (CAR-T cell number: AS cell number).
- the results of Test Example 11 are shown.
- the vertical axis shows the Cell index that reflects the number of Kelly cells on the E-plate.
- the horizontal axis shows the elapsed time from the addition of the target cells.
- the results of Test Example 12 are shown.
- the vertical axis shows the Cell index that reflects the number of SK-N-SH cells on the E-plate.
- the horizontal axis shows the elapsed time from the addition of the target cells.
- the results of Test Example 13 are shown.
- the vertical axis shows the Cell index that reflects the number of Hs578T-Luc cells on the E-plate.
- the horizontal axis shows the elapsed time from the addition of the target cells.
- the results of Test Example 14 are shown.
- the vertical axis shows the Cell index that reflects the number of BT549-Luc cells on the E-plate.
- the horizontal axis shows the elapsed time from the addition of the target cells.
- the results of Test Example 15 are shown.
- the vertical axis shows the Cell index that reflects the number of Kelly cells on the E-plate.
- the horizontal axis shows the elapsed time from the addition of the target cells.
- the results of Test Example 16 are shown.
- the vertical axis shows the Cell index that reflects the number of D8 cells on the E-plate.
- the horizontal axis shows the elapsed time from the addition of the target cells.
- the results of Test Example 17 are shown.
- the vertical axis shows the Cell index that reflects the number of C2 cells on the E-plate.
- the horizontal axis shows the elapsed time from the addition of the target cells.
- the results of Test Example 18 are shown.
- the vertical axis shows the Cell index that reflects the number of NCI-N417 cells on the E-plate.
- the horizontal axis shows the passage of time.
- Identity of amino acid sequences refers to the degree of agreement between two or more contrastable amino acid sequences with respect to each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences.
- the level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters.
- FASTA a sequence analysis tool
- the algorithm BLAST by Karlin and Altschul Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA.
- conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
- substitution between amino acid residues having a basic side chain such as lysine, arginine, and histidine is a conservative substitution.
- amino acid residues with acidic side chains such as aspartic acid and glutamic acid
- amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine
- Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan
- amino acid residues with beta-branched side chains such as threonine, valine and isoleucine
- aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitution between amino acid residues is also a conservative substitution.
- CDR is an abbreviation of C omplementarity D etermining R egion, also called complementarity determining regions.
- the CDR is a region existing in the variable region of immunoglobulin, and is a region deeply involved in the specific binding of the antibody to the antigen.
- the "light chain CDR” means a CDR existing in the light chain variable region of immunoglobulin
- the “heavy chain CDR” means a CDR existing in the heavy chain variable region of immunoglobulin.
- the "variable region” means an region including CDR1 to CDR3 (hereinafter, simply referred to as "CDRs1-3").
- CDRs1-3 CDR1 to CDR3
- the arrangement order of these CDRs1-3 is not particularly limited, but preferably, the frames are continuous or described later in the order of CDR1, CDR2, and CDR3 from the N-terminal side to the C-terminal side, or vice versa. It means a region arranged via another amino acid sequence called a work region (FR).
- the "heavy chain variable region” is a region in which the above-mentioned heavy chain CDRs1-3 is arranged, and the “light chain variable region” is a region in which the above-mentioned light chain CDRs1-3 is arranged.
- FR framework areas
- the region between the N-terminal of the variable region and CDR1 is FR1
- the region between CDR1 and CDR2 is FR2
- the region between CDR2 and CDR3 is FR3
- the region between CDR3 and the C-terminal of the variable region is Defined as FR4 respectively.
- the heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1 the heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3
- a heavy chain variable region comprising a heavy chain CDR3 comprising, and / or a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and represented by SEQ ID NO: 11.
- GD2-binding molecule (sometimes referred to herein as "the GD2-binding molecule of the invention") comprising a light chain variable region comprising a light chain CDR3 comprising an amino acid sequence. This will be described below.
- the GD2-binding molecule of the present invention is a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain containing the amino acid sequence shown in SEQ ID NO: 3.
- light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 10 and the amino acid sequence set forth in SEQ ID NO: 11. It is not particularly limited as long as it contains a light chain variable region containing the light chain CDR3 and is a molecule having binding to GD2.
- the GD2-binding molecule of the present invention may be a molecule composed of one kind of polypeptide or a molecule composed of a complex of two or more kinds of polypeptides. Further, the GD2-binding molecule of the present invention may be a molecule composed of a polypeptide or a complex thereof, or another substance (for example, a fluorescent substance, a radioactive substance, an inorganic particle, etc.) may be added to the polypeptide or the complex thereof. May be connected.
- the binding property to GD2 can be measured according to a known method, for example, by the ELISA method (specifically, by the method of Test Example 2, for example).
- the binding property of the GD2-binding molecule of the present invention to GD2 is, for example, 20% or more, 50% or more, 70% or more, 80 with respect to 100% binding to GD2 of the 220-51 antibody of the examples described later. % Or more, 90% or more, 95% or more, 99% or more.
- the GD2-binding molecule of the present invention preferably contains both the heavy chain variable region and the light chain variable region.
- the heavy chain variable region is preferably 90% or more (preferably 95% or more, preferably 98% or more, preferably 99%) of the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4. It is a heavy chain variable region containing an amino acid sequence having the same identity as above).
- the light chain variable region is preferably 90% or more (preferably 95% or more, preferably 98% or more, preferably 99%) with respect to the amino acid sequence shown by SEQ ID NO: 12 or the amino acid sequence shown by SEQ ID NO: 12. It is a light chain variable region containing an amino acid sequence having the same identity as above). If there is an amino acid mutation from SEQ ID NO: 4 or 12, the mutation is preferably an amino acid substitution, more preferably a conservative substitution of an amino acid.
- the GD2-binding molecule of the present invention can specifically recognize ganglioside GD2.
- the GD2-binding molecule of the present invention is at least one selected from the group consisting of ganglioside GD1a, ganglioside GD1b, ganglioside GD3, ganglioside GM1, ganglioside GM3, ganglioside GT1b, and lactosylceramide (preferably 2).
- Species or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 (all) binding to other antigens is 1/2 or less (preferably 1 /) of ganglioside GD2 binding 5 or less, 1/10 or less, 1/20 or less, 1/100 or less, 1/500 or less, 1/2000 or less, 1/10000 or less) is preferable.
- the GD2-binding molecule of the present invention may be chemically modified.
- R in the ester is, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; for example, phenyl.
- C 6-12 aryl groups such as ⁇ -naphthyl; for example, phenyl-C 1-2 alkyl groups such as benzyl, phenethyl; C 7- such as ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl. 14 Alkyl group; Pivaloyloxymethyl group etc. are used.
- the polypeptide constituting the GD2-binding molecule of the present invention may have a carboxyl group (or carboxylate) other than the C-terminal amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
- the polypeptide constituting the GD2 binding molecules of the present invention the amino group of the amino acid residues of the N-terminal protecting group (e.g., formyl group, such as C 1-6 alkanoyl such as acetyl group C 1-6 Those protected with an acyl group (such as an acyl group), those in which the N-terminal glutamine residue that can be cleaved and produced in vivo is pyroglutamine-oxidized, and substituents on the side chain of amino acids in the molecule (eg-OH,- SH, amino group, imidazole group, indol group, guanidino group, etc.) are protected by suitable protective groups (eg, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group, acetyl group) What is included is also included.
- suitable protective groups eg, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group, acet
- the GD2-binding molecule of the present invention may be one to which a protein or peptide such as a known protein tag or signal sequence is added.
- protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, fluorescent protein tag and the like.
- the GD2-binding molecule of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base.
- the salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and either an acidic salt or a basic salt can be adopted.
- acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, and apple.
- Organic acid salts such as acid salts, citrates, methane sulfonates and paratoluene sulfonates; amino acid salts such as asparaginates and glutamates can be mentioned.
- basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt.
- the GD2-binding molecule of the present invention may be in the form of a solvate.
- the solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid and the like.
- the GD2-binding molecule of the present invention is, in a preferred embodiment, an antibody (in the present specification, the GD2-binding molecule of the present invention, which is an antibody, may be referred to as "the antibody of the present invention").
- the antibody of the present invention is a monoclonal antibody.
- the molecular weight of the antibody of the present invention is not particularly limited, but the lower limit is, for example, 20,000, preferably 50,000, preferably 100,000, more preferably 120,000, and the upper limit is, for example, 1,000,000, preferably 500,000, more preferably 200,000. ..
- the structure of the antibody of the present invention is not particularly limited.
- the antibody of the present invention may contain a constant region or may not contain a constant region.
- the constant region of the heavy chain (CH1, CH2, and CH3) and the constant region of the light chain (CL) may be all included, and any one or more of them may be included. May include a combination of.
- immunoglobulin examples include immunoglobulin, Fab, F (ab') 2 , minibody, scFv-Fc, Fv, scFv, diabody, triabody, Examples include a tetrabody.
- immunoglobulin is preferably mentioned from the viewpoint of the effect of the present invention.
- Immunoglobulin has a structure in which two structures consisting of one heavy chain having a heavy chain variable region and a heavy chain constant region and one light chain having a light chain variable region and a light chain constant region are combined.
- Fab includes a heavy chain fragment containing CH1 in the heavy chain variable region and the heavy chain constant region, and a light chain containing the light chain variable region and the light chain constant region (CL), and the heavy chain variable region and the light chain. It has a structure in which the chain variable region is associated by the above-mentioned non-covalent intermolecular interaction or is bound by a disulfide bond.
- CH1 and CL may be disulfide-bonded with each other by the thiol groups of the cysteine residues present in each.
- F (ab') 2 has two pairs of the above Fabs, and CH1 is a structure formed by disulfide bonds between thiol groups of cysteine residues contained therein.
- a minibody is a structure in which two fragments in which CH3 is bound to the heavy chain variable region constituting the following scFv are associated with each other by non-covalent intermolecular interaction.
- scFv-Fc With scFv-Fc, the following two antibody fragments containing scFv, CH2, and CH3 are associated with each other by non-covalent intermolecular interaction between CH3s as in the case of the above minibody, and the cysteine residue contained in each CH3. It is a structure in which thiol groups of groups are disulfide-bonded to each other.
- Fv is also called the smallest structural unit of an antibody, and is a structure in which a heavy chain variable region and a light chain variable region are associated by a non-covalent intermolecular interaction.
- the thiol groups of cysteine residues existing in the heavy chain variable region and the light chain variable region may be disulfide-bonded to each other.
- scFv is a structure in which the C-terminal of the heavy chain variable region and the N-terminal of the light chain variable region are linked by a linker, or the N-terminal of the heavy chain variable region and the C-terminal of the light chain variable region are connected by a linker. It is a structure and is also called a single chain antibody.
- the above scFv forms a dimer, a trimer, and a tetramer, respectively, and like Fv, non-covalent intermolecular interactions between variable regions, etc.
- the structure is structurally stable.
- the antibody of the present invention is immunoglobulin
- its class is not particularly limited.
- the class include IgA, IgD, IgE, IgG, IgM, and the like, as well as subclasses thereof.
- Preferred classes include, for example, IgG, IgM, etc., preferably IgG, and more preferably IgG1.
- the origin of the antibody of the present invention is not particularly limited.
- the antibody of the present invention can be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, a chimpanzee-derived antibody, or the like.
- the antibody of the present invention includes chimeric antibodies (for example, antibodies obtained by replacing the amino acid sequence of the constant region of an antibody derived from a non-human organism (such as mouse) with the amino acid sequence of the constant region of a human-derived antibody), a humanized antibody, and the like. It may be a fully humanized antibody or the like.
- the antibody of the present invention can be produced, for example, by a method including a step of culturing a host transformed with a polynucleotide encoding the antibody of the present invention and recovering a fraction containing the antibody of the present invention.
- the polynucleotide encoding the antibody of the present invention is not particularly limited as long as it contains the antibody of the present invention in an expressible state, and may contain other sequences in addition to the coding sequence of the antibody of the present invention. Examples of other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, a replication base point, a drug resistance gene coding sequence, etc., which are arranged adjacent to the antibody coding sequence of the present invention. Be done. Further, the polynucleotide encoding the antibody of the present invention may be a linear polynucleotide or a cyclic polynucleotide (vector or the like).
- polynucleotide examples include (I) a polynucleotide containing a base sequence encoding at least one selected from the group consisting of heavy chains, heavy chain variable regions, and heavy chain CDRs1-3 of the antibody of the present invention. (II) A polynucleotide containing a base sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region, and the light chain CDRs1-3 of the antibody of the present invention, (III) the antibody of the present invention.
- Examples thereof include polynucleotides containing a base sequence encoding at least one selected from the group consisting of chain CDRs1-3.
- the host is not particularly limited, and examples thereof include insect cells, eukaryotic cells, and mammalian cells. Of these, mammalian cells such as HEK cells, CHO cells, NS0 cells, SP2 / O cells, and P3U1 cells are preferable from the viewpoint of more efficiently expressing the antibody.
- mammalian cells such as HEK cells, CHO cells, NS0 cells, SP2 / O cells, and P3U1 cells are preferable from the viewpoint of more efficiently expressing the antibody.
- the method of transformation, culturing, and recovery is not particularly limited, and a known method in antibody production can be adopted. After recovery, the antibody of the present invention may be purified if necessary. Purification can be performed by known methods in antibody production, such as chromatography, dialysis and the like.
- the GD2-binding molecule of the present invention is, in a preferred embodiment, a chimeric antigen receptor (in the present specification, the GD2-binding molecule of the present invention which is a chimeric antigen receptor is referred to as "chimeric of the present invention”. It may also be referred to as "antigen receptor").
- a chimeric antigen receptor is a single-chain antibody (scFv) in which a light chain (VL) and a heavy chain (VH) of a monoclonal antibody variable region are bound in series, and the binding property to an antigen is typically obtained. It is a chimeric protein that has an N-terminal side as a responsible region and also has a T cell receptor (TCR) ⁇ chain on the C-terminal side. T cells that express CAR are called CAR-T cells.
- the region responsible for binding to the antigen is a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2.
- the linker connecting the heavy chain variable region and the light chain variable region is not particularly limited as long as the function as a chimeric antigen receptor is maintained, and is arbitrary.
- a GS linker typically, a linker containing a repeating sequence having GGGGS (SEQ ID NO: 41) as a constituent unit
- the number of amino acid residues in the linker is, for example, 5 to 30, preferably 10 to 20, and more preferably 15.
- the chimeric antigen receptor of the present invention usually includes a scFv domain containing a heavy chain variable region and a light chain variable region, a transmembrane domain, and a core region containing an intracellular domain of TCR.
- the scFv domain, the transmembrane domain, and the intracellular domain of TCR are usually arranged in order from the N-terminal side, either directly or via other domains.
- transmembrane domain is not limited unless it inhibits the function of the chimeric antigen receptor.
- CD28, CD3zeta, CD4, CD8alpha and the like expressed in T cells and the like can be used. Mutations may be appropriately introduced into these transmembrane domains as long as they do not inhibit the function of the chimeric antigen receptor.
- the intracellular domain of TCR can be, for example, an intracellular domain derived from CD3, which is also called TCR ⁇ chain.
- a mutation may be appropriately introduced into CD3 as long as it does not inhibit the function of the chimeric antigen receptor.
- ITAM immunophilicity-associated activation motif
- the chimeric antigen receptor of the present invention preferably has a spacer sequence arranged between the scFv domain and the transmembrane domain.
- the length of the spacer sequence and the types of amino acid residues that compose it are not limited as long as they do not interfere with the function of the chimeric antigen receptor.
- the spacer sequence can be designed to have about 10 to 200 amino acid residues.
- the core region further contains the intracellular domain of the co-stimulator.
- the intracellular domain of the co-stimulator is not particularly limited as long as it is an intracellular domain derived from the co-stimulator possessed by T cells or the like.
- one or more selected from the group consisting of OX40, 4-1BB, GITR, CD27, CD278, CD28 and the like can be appropriately selected and used. Mutations may be appropriately introduced into the intracellular domains of these co-stimulators as long as they do not inhibit the function of the chimeric antigen receptor.
- the position of the intracellular domain of the co-stimulator is not particularly limited as long as it is on the C-terminal side of the transmembrane domain, and may be either the N-terminal side or the C-terminal side of the intracellular domain of TCR.
- the chimeric antigen receptor of the present invention preferably contains various ligand domains such as GITRL domain, 4-1BBL domain, and ICOSL domain via a self-cleaving peptide domain on the C-terminal side of the core region. This makes it possible to further enhance the expression efficiency of the chimeric antigen receptor and the cytotoxic activity of CAR-T cells containing the chimeric antigen receptor.
- self-cleaving peptide means a peptide sequence having a cleavage activity that occurs between two amino acid residues in the peptide sequence itself.
- examples of the self-cleaving peptide include a 2A peptide and a 2A-like peptide.
- cleavage occurs between glycine and proline residues on these peptides. This is caused by the "ribosome skipping mechanism", which prevents the formation of normal peptide bonds between glycine residues and proline residues during translation, and does not affect downstream translation.
- the ribosome skip mechanism is known in the art and is used for the expression of multiple proteins encoded by a single molecule of messenger RNA (mRNA).
- the self-cleaving peptide used in the present invention can be obtained from a viral 2A peptide or a 2A-like peptide having an equivalent function.
- 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV)
- F2A foot-and-mouth disease virus
- E2A E2A
- E2A horse rhinitis A virus
- P2A 2A peptide
- PTV-1 Porcine teschovirus
- TaV Thosea signavirus
- the GITRL domain is not particularly limited, but is 90% or more (preferably 95% or more, preferably 98% or more, preferably 98% or more, preferably 95% or more, preferably 98% or more, preferably 95% or more, preferably 98% or more, preferably 95% or more, preferably 98% or more, preferably 95% or more, preferably 98% or more, preferably the amino acid sequence shown by SEQ ID NO: 40 or the amino acid sequence shown by SEQ ID NO: 40.
- a domain containing an amino acid sequence having 99% or more) identity is preferred. If there is an amino acid mutation from SEQ ID NO: 40, the mutation is preferably an amino acid substitution, more preferably a conservative substitution of an amino acid.
- the technology for producing a chimeric antigen receptor and CAR-T cells expressing it is known. These can be produced according to or according to known methods.
- polynucleotides of the invention that encode the GD2-binding molecules of the invention. This will be described below.
- the polynucleotide of the present invention may contain other sequences in addition to the coding sequence of the GD2-binding molecule of the present invention.
- the polynucleotide of the present invention preferably contains the GD2-binding molecule of the present invention in an expressible state. Examples of other sequences include promoter sequences, enhancer sequences, repressor sequences, insulator sequences, replication base points, reporter protein (for example, fluorescent protein, etc.) coding sequences, drug resistance gene coding sequences, and the like.
- the polynucleotide of the present invention may be a linear polynucleotide or a cyclic polynucleotide (vector or the like).
- the vector can be a plasmid vector or a viral vector (eg, adenovirus, or retrovirus).
- the vector can be, for example, a cloning vector or an expression vector.
- the expression vector include a vector for prokaryotic cells such as Escherichia coli and actinomycetes, and a vector for eukaryotic cells such as yeast cells, insect cells, and mammalian cells.
- the polynucleotide of the present invention includes not only DNA and RNA, but also those to which known chemical modifications are applied, as illustrated below.
- PS phosphorothioate
- methylphosphonate methylphosphonate
- phosphorodithionate to prevent degradation by hydrolases such as nucleases.
- the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is changed to -OR (R is, for example, CH3 (2'-O-Me), CH 2 CH 2 OCH 3 (2'-O-MOE), CH.
- the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl.
- examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with a biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto.
- polynucleotide includes not only natural nucleic acids but also any of BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid) and the like.
- the present invention relates to a cell (sometimes referred to as "the cell of the present invention” in the present specification) containing the polynucleotide of the present invention. This will be described below.
- the cell from which the cell of the present invention is derived is not particularly limited.
- the derived cells include cells that can be used for protein expression (for example, insect cells, eukaryotic cells, mammalian cells, etc.). Can be mentioned.
- the cell of the present invention contains a polynucleotide encoding the chimeric antigen receptor of the present invention
- the cell is preferably a T cell.
- the T cell is preferably a cell expressing the chimeric antigen receptor of the present invention, and in a more specific embodiment, the T cell expresses the chimeric antigen receptor of the present invention on the cell membrane.
- the chimeric antigen receptor of the present invention is expressed with the GD2-binding region exposed outside the cell membrane.
- T cells expressing the chimeric antigen receptor After recognizing GD2 in the GD2-binding region, T cells expressing the chimeric antigen receptor transmit the recognition signal to the inside of the T cells, etc., and activate a signal that induces cytotoxic activity, which is linked to this. The cell exerts an aggression or cytotoxic activity against other cells or tissues expressing GD2.
- CAR-T cell When the cell that exerts such a function is CTL, it is called a chimeric antigen receptor T cell (CAR-T cell). Similar to chimeric antigen receptor T cells, cells having the potential to exert cytotoxic activity such as NK cells can also exert cytotoxic activity by binding the GD2-binding region to GD2. Therefore, a host cell containing a polynucleotide encoding a chimeric antigen receptor (particularly, a host cell having cytotoxic activity) is useful as an active ingredient of a pharmaceutical composition.
- CAR-T cells and the like specifically recognize cancer tissues (tumor tissues), they are useful for the treatment or prevention of cancers and the like.
- the type of cancer is not particularly limited and includes solid cancer and blood cancer.
- solid cancers include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumor, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, and osteosarcoma. , Chondrosarcoma, rhabdomyosarcoma, melanoma, neuroblastoma, bladder cancer and the like.
- the cell of the present invention can be obtained by introducing the polynucleotide of the present invention into the cell. If necessary, cells containing the polynucleotide of the present invention may be concentrated, or a specific marker (CD antigen such as CD8) may be used as an index for concentration.
- a specific marker CD antigen such as CD8
- composition in one aspect of the present invention, a chimeric antigen receptor T cell or a chimeric antigen receptor NK cell containing a polynucleotide encoding the chimeric antigen receptor of the present invention, or an antibody of the present invention is contained. It relates to a pharmaceutical composition (in the present specification, it may be referred to as "the pharmaceutical composition of the present invention"). This will be described below.
- the content of the above-mentioned cells and antibodies in the pharmaceutical composition takes into consideration the type of the target disease (for example, solid cancer), the target therapeutic effect, the administration method, the treatment period, the age of the patient, the weight of the patient, and the like. Can be set as appropriate.
- the content of the antibody in the pharmaceutical composition can be about 0.001 part by weight to 10 parts by weight, assuming that the entire pharmaceutical composition is 100 parts by weight.
- the content of cells in the pharmaceutical composition can be, for example, about 1 cell / mL to 10 ⁇ 4 cells / mL.
- the administration form of the pharmaceutical composition is not particularly limited as long as the desired effect can be obtained, and either oral administration or parenteral administration (for example, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, local administration). It can be administered to mammals including humans by the same route of administration. Since the active ingredient is a cell, the preferred dosage form is parenteral administration, more preferably intravenous injection. Dosage forms for oral administration and parenteral administration and methods for producing the same are well known to those skilled in the art, and the antibody or cells according to the present invention are mixed with a pharmaceutically acceptable carrier or the like according to a conventional method. Can be manufactured.
- the dosage forms for parenteral administration are injectable preparations (eg, infusions, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (eg, ointments, suppositories, lotions). Agents), suppository inhalants, eye agents, eye ointments, nasal drops, ear drops, liposomes and the like.
- injectable preparations are prepared by dissolving or suspending antibodies or cells in distilled water for injection, and if necessary, lysis aids, buffers, pH regulators, isotonic agents, soothing agents, preservatives. , And stabilizers and the like can be added.
- the pharmaceutical composition can also be a lyophilized preparation for preparation at the time of use.
- the pharmaceutical composition may further contain other agents effective in treating or preventing the disease.
- the pharmaceutical composition may contain components such as a bactericidal agent, an anti-inflammatory agent, a cell activator, vitamins, and amino acids, if necessary.
- Carriers used in the formulation of pharmaceutical compositions include excipients, binders, disintegrants, lubricants, colorants, flavoring agents commonly used in the art and, if necessary, stabilizers, emulsifiers and absorptions.
- Use accelerators, surfactants, pH regulators, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, etc. Can be done.
- the type of disease to be treated or prevented using the pharmaceutical composition is not particularly limited as long as the treatment or prevention can be achieved.
- Specific target diseases include, for example, tumors.
- the tumor is preferably a GD2-positive tumor.
- the type of tumor is not particularly limited and includes solid cancer and hematological cancer. Examples of solid cancers include lung cancer (particularly small cell lung cancer), colon cancer, ovarian cancer, breast cancer, brain tumor, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, and chondrosarcoma. , Rhabdomyosarcoma, melanoma, neuroblastoma, bladder cancer and the like.
- the administration target (subject) of the pharmaceutical composition is, for example, an animal suffering from or a possibility of suffering from the above-mentioned disease. "May be affected" can be determined by a known diagnostic method.
- the animal is, for example, a mammal, preferably a human.
- the dose of the pharmaceutical composition is, for example, the route of administration, the type of disease, the degree of symptoms, the age, sex, weight, the severity of the disease, the pharmacokinetic and toxicological characteristics of the patient, and the drug. It can be determined by the clinician based on various factors such as the availability of a delivery system and whether it is administered as part of a combination of other drugs.
- the dose of the pharmaceutical composition can be, for example, about 1 microgram / kg (body weight) to 10 g / kg (body weight) per day when the active ingredient is an antibody. When the active ingredient is cells, it can be about 10 ⁇ 4 cells / kg (body weight) to 10 ⁇ 9 cells / kg (body weight).
- the administration schedule of the pharmaceutical composition can also be determined in consideration of the same factors as the dose thereof. For example, the above daily dose can be administered once a day to January.
- GD2-expressing cells S1 and S6 were introduced by incorporating GD3 synthase and GM2 / GD2 synthase cDNA into pCDNA3.1 neo into the sub-strain N1 (GD3, GD3 not expressed) of SK-MEL-28 cells.
- V4 and V9 were introduced with the empty vector pCDNA3.1neo.
- APC / Cy7 labeled anti-human CD8 antibody (clone HT8a) was purchased from Biolegend.
- the FITC-labeled anti-human Vd2 antibody (clone B6, 331418) was purchased from Biolegeng.
- V450-labeled anti-human IFNg antibody (clone 45.83, 48-7319-42) purchased from BD Pharmingen.
- PE / Cy7 labeled anti-human TNFa antibody (clone Mab11, 12-7349-82) was purchased from eBioscience.
- APC-labeled anti-human CD107a antibody (560664) was purchased from BD Pharmingen.
- PBMC peripheral blood mononuclear cells adjusted by Ficoll were added to 0.6% human plasma and final concentration.
- the cells were cultured in GT-T551 supplemented with 600 u / ml IL-2, recovered at 4 days of day, and infected with a retrovirus immobilized at 42 ° C for 2 hours at 2000 xg for 2 hours and cultured. ..
- the gamma / delta cells were prepared according to the method of Tanaka et al.
- Gamma / delta cells peripheral blood mononuclear cells were cultured in YM-AB containing a novel bisphosphonate preparation (PTA), IL-7 and IL-15 were added at 25 ng / ml each, and recovered on day 4. ) was infected, and the cells were cultured in the same medium.
- PTA novel bisphosphonate preparation
- IL-7 and IL-15 were added at 25 ng / ml each, and recovered on day 4.
- CAR and GITRL expression Regarding CAR expression, anti-kappa antibody was reacted at 10 microgram / ml, washed, and Alexa488-labeled anti-rabbit IgG (Invitrogen) was reacted at 5 microgram / ml. After washing, staining with APC / Cy7-labeled anti-human CD8 (BD) and APC-labeled anti-human CD4 antibody (Biolegend) was performed, and measurement was performed with FACS CANT. Regarding the expression of GITRL, PE-labeled anti-human GITRL (Biolegend) was reacted at 100-fold dilution and measured by FACS CANT.
- PE-labeled anti-human GITRL antibody Biolegend was used for staining with BD Cytofix / Cytoperm and BD Perm / Wash. The method followed the manufacturer's instructions.
- the target cells AS 1.5x10 ⁇ 4 suspended in RPMI1640 10% FCS 100 micro litter were placed and allowed to stand in a CO 2 incubator for 24 hours, and then suspended in RPMI1640 10% FCS 100 microliter. Effector cells 1.5x10 ⁇ 4 were inserted and subsequent current changes were recorded.
- Non-RI cytotoxicity measurement The experimental method followed the manufacturer's instructions. That is, first, the target AS cells 4x10 ⁇ 5 cells were suspended in 10% FCS / PRMI1640 of 400 micro litter, 1 micro litter of BM-HT solution was added thereto, and the cells were cultured in a CO2 incubator for 15 minutes and washed. After that, 5x10 ⁇ 3 cells were prepared, 50x10 ⁇ 3, 15x10 ⁇ 3, 5x10 ⁇ 3 CAR T cells were placed therein, co-cultured for 2 hours, and then centrifuged to collect the supernatant of 25 micro litter. .. After adding EU solution 250 micro litter to this and mixing, the luminescence was measured with TriStar2 SLB942 Multimode Reader (Berthold Technologies).
- Test example 1 Isolation of Monoclonal Antibodies Balb / cx C57BL / 6 F1 mice were immunized by three subcutaneous inoculations of IMR32 cells, and the collected spleen cells were fused with NS-1 cells and cultured in RPMI1640 medium containing 10% FCS and HAT. Then, a monoclonal antibody was prepared. The obtained antibody was screened by flow cytometry recognition on IMR32 cells, and further subclones were obtained for the obtained clone 220 to obtain 220-51.
- Test example 2 Analysis of antigen specificity 1
- the antigen specificity of the 220-51 antibody was analyzed by ELISA.
- Ganglioside GD1a, GD1b, GD2, GD3, GM1, GM3, GT1b and lactosylceramide were immobilized in methanol at 50 ng each, and ascites antibody at each dilution stage was allowed to act on the HRP-labeled anti-mouse IgG antibody (manufactured by Southern Biotech). Was reacted, color was developed by OPD, and the absorbance was measured.
- the results are shown in Fig. 1.
- the 220-51 antibody recognized only GD2 and not other gangliosides.
- Test example 3 Analysis of antigen specificity 2
- the antigen specificity of the 220-51 antibody was analyzed by thin layer chromatography.
- a mixture of bovine-derived ganglioside and GM3, and cancer cells SK-MEL-23 (Carney2) and AS-derived ganglioside were developed by thin-layer chromatography and heated blotter (ATTO TLC Thermal Blotter AC5970, Atto, Tokyo).
- 220-51 antibody was allowed to act.
- HRP-labeled anti-mouse IgG whole
- Cell Signaling was allowed to act on the secondary antibody, and the light was emitted by Western LIGHTNING TM Plus ECL (Perkin Elmer Inc., Waltham, MA).
- the 220-51 antibody recognized only GD2 and not other gangliosides.
- Test example 4 Recognition of GD2-expressing cells 1x10 ⁇ 5 cells were treated with an antibody diluted 100-fold with 0.5% BSA / PBS at room temperature for 30 minutes, washed, and treated with FITC-labeled anti-mouse IgG antibody (manufactured by Cappel). After washing with PBS, the measurement was performed with FACS Caliver or Accuri C6.
- Test example 5 GD2 + melanoma S1, S6 cells and GD2-V4, V9 cells (cell number 1x10 ⁇ 4) were sown on a plate on which cell adhesion-inhibiting collagen was immobilized, and 220-51 antibody was applied for 0.5 hours and 3 hours. Adhesion was observed by RT-CES when allowed to act at 50-fold dilution (S1-T: antibody added to S1).
- Test example 6 Sequence analysis The amino acid sequence of the 220-51 antibody and the base sequence encoding the antibody were analyzed. The analysis results are shown below. The CDR sequence was estimated by IMGIT.
- Heavy chain CDR1 amino acid sequence GFSLPSYG (SEQ ID NO: 1) Heavy chain CDR2 amino acid sequence: IWAGGITN (SEQ ID NO: 2) Heavy chain CDR3 amino acid sequence: ARGGSDYDGFAY (SEQ ID NO: 3) Heavy chain variable part amino acid sequence: EVQLVESGPGLVAPSQSLSITCTVSGFSLPSYGVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLTISKDNSKSQVFLKMNSLQTDDTAIYYCARGGSDYDGFAYWGQGTLVTVS (SEQ ID NO: 4) Heavy chain CDR1 base sequence: GGG TTT TCA TTA CCC AGC TAT GGT (SEQ ID NO: 5) Heavy chain CDR2 base sequence: ATC TGG GCT GGT GGA ATC ACA AAT (SEQ ID NO: 6) Heavy chain CDR3 base sequence: GCC AGA GGC GGC TCT GAT TAC GAC GGC TTT GCT TAC (SEQ ID NO:
- Light chain CDR1 amino acid sequence QSLLSSRTRKNY (SEQ ID NO: 9)
- Light chain CDR2 amino acid sequence WAS (SEQ ID NO: 10)
- Light chain CDR3 amino acid sequence KQSYNLRT (SEQ ID NO: 11)
- Light chain variable part amino acid sequence DIVMTQSPSSLAVSAGEKVTMNCRSSQSLLSSRTRKNYLAWYQQKPGQSPKLLIYWASIRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLRTFGGGTKLEIK (SEQ ID NO: 12)
- Light chain CDR1 base sequence CAG AGT CTC CTC AGC AGT AGA ACC CGA AAG AAC TAC (SEQ ID NO: 13)
- Light chain CDR2 base sequence TGG GCA TCT (SEQ ID NO: 14)
- Light chain CDR3 base sequence AAG CAA TCT TAT AAT CTT CGG ACG (SEQ ID NO: 15)
- Test example 7 Construction of CAR expression plasmid
- CAR expression plasmid Four types of CAR (28z CAR, zG CAR, 28z GITRL CAR, zG GITRL CAR) using the amino acid sequence of 220-51 antibody were designed (schematic diagram of the structure is shown in FIG. 5).
- CAR expression plasmid was prepared. Specifically, it was produced as follows.
- P2A-GITRL amino acid sequences are as follows: GSGATNFSLLKQAGDVEENPGP (P2A amino acid sequence: SEQ ID NO: 39) -MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS (GITRL amino acid sequence: SEQ ID NO: 40).
- an expression plasmid in which the P2A-GITRL base sequence is integrated is prepared adjacent to the 3'side of the base sequence of SEQ ID NO: 35 of the zGCAR expression plasmid using an artificial gene and PCR. did.
- Test example 8 Introduction of CAR gene into T cells and confirmation of expression
- the plasmid DNA constructed above was introduced into Plat-A cells to prepare a retrovirus, which was then infected with cultured human PBMC to obtain CAR-introduced T cells.
- CAR expression was examined by flow cytometry. Expression of CAR and ligand was confirmed in alpha / beta T cells as shown in FIGS. 6 to 11 and in gamma / delta T cells as shown in FIGS. 12 to 13. This is an effector cell.
- the expression efficiency and expression intensity of CAR (mean fluorescent intensity, indicated by MFI) were enhanced by co-expression of GITRL (Figs. 6 to 8).
- Test example 9 Recognition of target cells by effector cells CAR T cells are activated when co-cultured with target AS cells and express IFNg and TNFa. In addition, CD107a translocates to the cell surface. These reactions indicate that a multifunctional reaction has occurred. It was confirmed that all the CAR T cells produced this time were activated by co-culture with AS cells, and these reactions occurred (Figs. 14 to 16).
- Test example 10 Confirmation of cytotoxic effect by effector cells The cytotoxic effect of effector cells on AS cells was examined by temporal changes by xCelligence. As a result, it was shown that GD2 CAR has sufficient cytotoxic activity even in alpha / beta T cells and gamma / delta T cells (FIGS. 17 and 18). This was also observed in non-RI cytotoxicity studies (Fig. 19).
- Test example 11 Analysis of cytotoxic effects by effector cells 1 After culturing 20000 GD2-positive Kelly cells on an E-plate for 20 hours, 40,000 effector cells (alpha / beta) were added and cultured, and the cell index was followed over time.
- the Cell index reflects the number of Kelly cells on the E-plate.
- the Normalized cell index was standardized with the number of Kelly cells immediately before co-culturing with effector cells as 1.
- Effective cytotoxicity by GD2 28z CAR T cells co-expressed with GD2 28z, GD2 zG, and GITRL was observed, and no cytotoxicity by PBMC without CAR introduction was observed (Fig. 20).
- Test example 13 Analysis of cytotoxic effects by effector cells 3 After culturing 15,000 GD2-positive Hs578T-Luc cells on an E-plate for 20 hours, 40000 effector cells (alpha / beta) were allowed to act on the cells, and the cell index was followed over time.
- the Cell index reflects the number of Hs578T-Luc cells on the E-plate.
- the Normalized cell index was standardized with the number of Hs578T-Luc cells immediately before co-culturing with effector cells as 1.
- Test example 14 Analysis of cytotoxic effects by effector cells 4 After culturing 20000 GD2-negative BT549-Luc cells on an E-plate for 18 hours, 40000 effector cells (alpha / beta) were allowed to act on the cells, and the cell index was followed over time.
- the Cell index reflects the number of BT549-Luc cells on the E-plate.
- the Normalized cell index was standardized with the number of BT549-Luc cells immediately before co-culturing with effector cells as 1.
- Test example 18 Analysis of cytotoxic effects by effector cells 7 After culturing 20000 GD2-positive NCI-N417 cells on an E-plate for 34 hours, 60000 effector cells (alpha / beta) were allowed to act on the cells, and the cell index was followed over time.
- the Cell index reflects the number of NCI-N417 cells on the E-plate.
- the Normalized cell index was standardized with the number of NCI-N417 cells immediately before co-culturing with effector cells as 1.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
Abstract
Description
本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。
本発明は、その一態様において、配列番号1で示されるアミノ酸配列を含む重鎖CDR1、配列番号2で示されるアミノ酸配列を含む重鎖CDR2、及び配列番号3で示されるアミノ酸配列を含む重鎖CDR3を含む重鎖可変領域、並びに/或いは配列番号9で示されるアミノ酸配列を含む軽鎖CDR1、配列番号10で示されるアミノ酸配列を含む軽鎖CDR2、及び配列番号11で示されるアミノ酸配列を含む軽鎖CDR3を含む軽鎖可変領域を含む、GD2結合性分子(本明細書において、「本発明のGD2結合性分子」と示すこともある。)に関する。以下に、これについて説明する。
本発明のGD2結合性分子は、好ましい一態様において、抗体である(本明細書において、抗体である本発明のGD2結合性分子について、「本発明の抗体」と示すこともある。)。
本発明のGD2結合性分子は、好ましい一態様において、キメラ抗原受容体である(本明細書において、キメラ抗原受容体である本発明のGD2結合性分子について、「本発明のキメラ抗原受容体」と示すこともある。)。
本発明は、その一態様において、本発明のGD2結合性分子をコードする、ポリヌクレオチド(本明細書において、「本発明のポリヌクレオチド」と示すこともある。)に関する。以下、これについて説明する。
本発明は、その一態様において、本発明のポリヌクレオチドを含有する、細胞(本明細書において、「本発明の細胞」と示すこともある。)に関する。以下、これについて説明する。
本発明は、その一態様において、本発明のキメラ抗原受容体をコードするポリヌクレオチドを含有するキメラ抗原受容体T細胞若しくはキメラ抗原受容体NK細胞、又は本発明の抗体を含有する、医薬組成物(本明細書において、「本発明の医薬組成物」と示すこともある。)に関する。以下、これについて説明する。
以下、特に断りの無い限り、各試験例では、以下の材料、方法を採用した。
Carney及びASはDr. Oldより得た。IMR32は、CEM, Kokl-3, MOLT4は (Dr. Old/ Ueda)。YTN17はYodoi 博士から、SK-MEL-28細胞の亜株N1は、Dr. Lloyd より供与された。NCI-417, ACC-LC-171, ACC-LC-96, ACC-LC-17は高橋隆博士から供与された。C-2細胞D-18はACC-LC-17にGD3合成酵素を導入して作成した。GD2発現細胞S1, S6はSK-MEL-28細胞の亜株N1(GD3, GD3無発現)にGD3合成酵素及びGM2/GD2合成酵素cDNAをpCDNA3.1neoに組み込んで導入した。また、空ベクターpCDNA3.1neoを導入したものがV4, V9である。
ウサギ抗ヒトkappa抗体(159)はMBLより購入した。Alexa488標識抗ウサギIgG抗体(A11034)はInvitrogenより購入した。PE標識抗GITRL抗体(FAB6941P)はBiolegendより購入した。PE標識抗ヒト4-1BB抗体(311504)はBiolegendより購入した。PE標識抗ヒトICOSL抗体(309404)はBiolegendより購入した。APC標識抗ヒトCD4抗体(clone RPA-T4)はInvitrogenより購入した。PE標識抗ヒトCD4抗体(555347)はBDより購入した。APC/Cy7標識抗ヒトCD8抗体(clone HT8a)はBiolegendより購入した。FITC標識抗ヒトVd2抗体(clone B6, 331418)はBiolegengより購入した。V450標識抗ヒトIFNg抗体(clone 45.83, 48-7319-42)BD Pharmingenより購入した。PE/Cy7標識抗ヒトTNFa抗体(clone Mab11, 12-7349-82)はeBioscienceより購入した。APC標識抗ヒトCD107a抗体(560664)はBD Pharmingenより購入した。
ユーロフィンに依頼して作製したCD1928及びCD1928z GITRLを制限酵素NotIとXhoIにて酵素処理し、pMS3に組み替えてプラスミドベクターを作製した。Luciferase NGFR発現ベクターについては、この二つをユーロフィンの受託合成によって作製し、それぞれNotIとClaI, ClaIとXhoIにて処理し、pMS3に組み替えて作製した。それらを用いてFUGENEを使用してPlatAに導入し、レトロウィルスを作製した。方法はメーカーの指示に従った。
OKT3 2 micro gramとレトロネクチン 10 micro gramを12 well plateに固相化したのち、フィコールによって調整した末梢血単核球を、0.6%ヒト血漿と終濃度600 u/mlのIL-2を加えたGT-T551にて培養し、day 4時点にて回収し、それに2000xg、2時間42℃にて固相化したレトロウィルスを感染して培養を行った。
gamma/delta細胞の作製は田中らの方法に従った。Gamma/delta細胞(末梢血単核球を新規ビスホスフォネート製剤(PTA)を含むYM-ABにて培養し、IL-7、IL-15をそれぞれ25 ng/ml添加し、day 4に回収)に感染させ、同培地にて培養を行った。
CARの発現に関しては、抗kappa抗体を10 micro gram /mlで反応させたのち洗浄し、Alexa488標識抗ウサギIgG(Invitrogen)を5 micro gram /mlで反応させて洗浄したのち、APC/Cy7標識抗ヒトCD8(BD)とAPC標識抗ヒトCD4抗体(Biolegend)による染色を行い、FACS CANTにて測定を行った。GITRLの発現に関してはPE標識抗ヒトGITRL(Biolegend)を100倍希釈にて反応させ、FACS CANTにて測定を行った。GITRLの細胞内染色に関してはPE標識抗ヒトGITRL抗体(Biolegend)を使用してBD Cytofix/Cytoperm及びBD Perm/Washによる染色を行った。方法はメーカーの指示に従った。
CAR遺伝子導入PBMCと標的細胞を混ぜ合わせたのちAPC標識抗ヒトCD107a抗体を反応させてCO2インキュベーターにて1時間培養したのちGolgi stopを作用させてCO2インキュベーターにて4時間培養し、洗浄したのち、抗ヒトkappa抗体及びAlexa488標識抗ウサギIgG抗体による染色を行ったのち、APC/Cy7標識抗CD8抗体及びPE標識抗ヒトCD4抗体による染色を行い、BD Cytofix/Cytoperm及びBD Perm/Washによる処理を行ったのちV450標識抗ヒトIFNg及びPE/Cy7標識抗ヒトTNFa染色を行った。
RPMI1640 10%FCS 100 micro litterに懸濁した標的細胞AS 1.5x10^4を入れてCO2培養器にて24時間静置したのち、RPMI1640 10%FCS 100 micro literに懸濁したエフェクター細胞1.5x10^4を入れてその後の電流変化を記録した。
実験方法はメーカーの指示に従った。即ち、まず、標的のAS細胞4x10^5細胞を400 micro litterの10%FCS/PRMI1640に懸濁し、これに1 micro litterのBM-HT溶液を加え、CO2インキュベーターにて15分培養し、洗浄したのち5x10^3細胞を準備し、これに50x10^3, 15x10^3, 5x10^3のCAR T細胞を入れ、2時間の共培養を行ったのち、遠心して25 micro litterの上清を回収した。これにEU solution 250 micro litterを加えて混ぜたのち、TriStar2 SLB942 Multimode Reader (Berthold Technologies社)にて発光の測定を行った。
Balb/c x C57BL/6 F1マウスを3回のIMR32細胞皮下接種によって免疫し、採取した脾臓細胞をNS-1細胞と融合させて10%FCS とHATを含むRPMI1640培地にて培養し、モノクローナル抗体を作製した。得られた抗体はIMR32細胞に対するフローサイトメトリ認識でスクリーンし、得られたクローン220についてさらにサブクローンを取得し、220-51を得た。
220-51抗体の抗原特異性をELISAで解析した。ガングリオシドGD1a, GD1b, GD2, GD3, GM1, GM3, GT1bおよびlactosylceramideをそれぞれ50 ngをメタノールにて固相化し、各希釈段階の腹水抗体を作用させ、HRP標識抗マウスIgG抗体(Southern Biotech社製)を反応させ、OPDによる発色を行い、吸光度を測定した。
220-51抗体の抗原特異性を薄層クロマトグラフィーで解析した。牛由来のgangliosideにGM3を混ぜたもの、およびがん細胞SK-MEL-23(Carney2), AS由来のgangliosideを薄層クロマトグラフィーで展開し、heat blotter (ATTO TLC Thermal Blotter AC5970, Atto, Tokyo) にてPVDF膜に転写したのち、220-51抗体を作用させた。その後、HRP標識抗マウス抗体secondary antibody, HRP-conjugated anti-mouse IgG (whole) (Cell Signaling)を作用させ、WESTERN LIGHTNINGTM Plus ECL (Perkin Elmer Inc., Waltham, MA)にて発光させた。
1x10^5の細胞を0.5% BSA/PBSにて100倍希釈した抗体で室温30分処理したのち、洗浄して、FITC標識抗マウスIgG抗体(Cappel社製)にて処理し、PBSで洗浄したのち、FACS CaliverまたはAccuriC6にて測定した。
コラーゲンを固相化したplateに対して、GD2+のメラノーマS1, S6細胞及びGD2-の V4, V9細胞(細胞数1x10^4)を蒔いて、220-51抗体を0.5時間、3時間で50倍希釈で作用させさせたときの接着をRT-CESによって観察した(S1-T: S1に抗体添加)。
220-51抗体のアミノ酸配列、及び該抗体をコードする塩基配列を解析した。解析結果を以下に示す。なお、CDRの配列はIMGITで推定した。
重鎖CDR1アミノ酸配列:GFSLPSYG(配列番号1)
重鎖CDR2アミノ酸配列:IWAGGITN(配列番号2)
重鎖CDR3アミノ酸配列:ARGGSDYDGFAY(配列番号3)
重鎖可変部アミノ酸配列:EVQLVESGPGLVAPSQSLSITCTVSGFSLPSYGVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLTISKDNSKSQVFLKMNSLQTDDTAIYYCARGGSDYDGFAYWGQGTLVTVS(配列番号4)
重鎖CDR1塩基配列:GGG TTT TCA TTA CCC AGC TAT GGT(配列番号5)
重鎖CDR2塩基配列:ATC TGG GCT GGT GGA ATC ACA AAT(配列番号6)
重鎖CDR3塩基配列:GCC AGA GGC GGC TCT GAT TAC GAC GGC TTT GCT TAC(配列番号7)
重鎖可変部塩基配列:GAGGTGCAGCTGGTGGAGTCTGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTACCCAGCTATGGTGTTCACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATCTGGGCTGGTGGAATCACAAATTATAACTCGGCTCTCATGTCCAGACTGACCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTTCAAACTGATGACACAGCCATATACTACTGTGCCAGAGGCGGCTCTGATTACGACGGCTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCATCA(配列番号8)。
軽鎖CDR1アミノ酸配列:QSLLSSRTRKNY(配列番号9)
軽鎖CDR2アミノ酸配列:WAS(配列番号10)
軽鎖CDR3アミノ酸配列:KQSYNLRT(配列番号11)
軽鎖可変部アミノ酸配列:DIVMTQSPSSLAVSAGEKVTMNCRSSQSLLSSRTRKNYLAWYQQKPGQSPKLLIYWASIRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLRTFGGGTKLEIK(配列番号12)
軽鎖CDR1塩基配列:CAG AGT CTC CTC AGC AGT AGA ACC CGA AAG AAC TAC(配列番号13)
軽鎖CDR2塩基配列:TGG GCA TCT(配列番号14)
軽鎖CDR3塩基配列:AAG CAA TCT TAT AAT CTT CGG ACG(配列番号15)
軽鎖可変部塩基配列:GACATTGTGATGACACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAACTGCAGATCCAGTCAGAGTCTCCTCAGCAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCTATTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(配列番号16)。
220-51抗体のアミノ酸配列を使用した4種のCAR(28z CAR、zG CAR、28z GITRL CAR、zG GITRL CAR)をデザインし(構造の模式図を図5に示す)、これらのCARの発現プラスミドを作製した。具体的には、次のようにして作製した。
NotI site kozak sequence:GCGGCCGCCACC(配列番号17)-
mVH leader:ATGAACTTTGGGCTCAGATTGATTTTCCTTGTCCTTACTTTAAAAGGTGTGAAGTGT(配列番号18)-
mVH:GAGGTGCAGCTGGTGGAGTCTGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTACCCAGCTATGGTGTTCACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATCTGGGCTGGTGGAATCACAAATTATAACTCGGCTCTCATGTCCAGACTGACCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTTCAAACTGATGACACAGCCATATACTACTGTGCCAGAGGCGGCTCTGATTACGACGGCTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCATCA(配列番号19)-
single chain:GGAGGTGGAGGTTCTGGTGGAGGAGGTTCAGGTGGAGGTGGATCA(配列番号20)-
mVkappa:GACATTGTGATGACACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAACTGCAGATCCAGTCAGAGTCTCCTCAGCAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCTATTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCT TAT AAT CTT CGG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC AAA (配列番号21)-
hCkappa:CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCGGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGCGCGCCA(配列番号22)-
hCD28 transmembrane:ACTAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG(配列番号23)-
hCD28 intracellular domain:AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(配列番号24)-
hCD3 zeta:CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(配列番号25)TAA-
XhoI site:TCGATTCTCGAG(配列番号26)
<28z CARアミノ酸配列>
mVH leader:MNFGLRLIFLVLTLKGVKC(配列番号27)-
mVH:EVQLVESGPGLVAPSQSLSITCTVSGFSLPSYGVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLTISKDNSKSQVFLKMNSLQTDDTAIYYCARGGSDYDGFAYWGQGTLVTVS(配列番号28)-
single chain:GGGGSGGGGSGGGGS(配列番号29)-
mVkappa:DIVMTQSPSSLAVSAGEKVTMNCRSSQSLLSSRTRKNYLAWYQQKPGQSPKLLIYWASIRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLRTFGGGTKLEIK(配列番号30)-
hCkappa:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGECGAP(配列番号31)-
hCD28 transmembrane:TRFWVLVVVGGVLACYSLLVTVAFIIFWVR(配列番号32)-
hCD28 intracellular domain:SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(配列番号33)-
hCD3 zeta:LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*(配列番号34)-
<zG CAR作製用人工遺伝子塩基配列>
NotI site kozak sequence:GCGGCCGCCACC(配列番号17)-
mVH leader:ATGAACTTTGGGCTCAGATTGATTTTCCTTGTCCTTACTTTAAAAGGTGTGAAGTGT(配列番号18)-
mVH:GAGGTGCAGCTGGTGGAGTCTGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTACCCAGCTATGGTGTTCACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATCTGGGCTGGTGGAATCACAAATTATAACTCGGCTCTCATGTCCAGACTGACCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTTCAAACTGATGACACAGCCATATACTACTGTGCCAGAGGCGGCTCTGATTACGACGGCTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCATCA(配列番号19)-
single chain:GGAGGTGGAGGTTCTGGTGGAGGAGGTTCAGGTGGAGGTGGATCA(配列番号20)-
mVkappa:GACATTGTGATGACACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAACTGCAGATCCAGTCAGAGTCTCCTCAGCAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCTATTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCT TAT AAT CTT CGG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC AAA (配列番号21)-
hCkappa:CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCGGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGCGCGCCA(配列番号22)-
hCD28 transmembrane:ACTAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG(配列番号23)-
hCD3 zeta:CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(配列番号25)-
hGITR intracellular domain:AGGAGTCAGTGCATGTGGCCCCGAGAGACCCAGCTGCTGCTGGAGGTGCCGCCGTCGACCGAAGACGCCAGAAGCTGCCAGTTCCCCGAGGAAGAGCGGGGCGAGCGATCGGCAGAGGAGAAGGGGCGGCTGGGAGACCTGTGGGTG(配列番号35)TAA-
XhoI site:TCGATTCTCGAG(配列番号26)
<zG CARアミノ酸配列>
mVH leader:MNFGLRLIFLVLTLKGVKC(配列番号27)-
mVH:EVQLVESGPGLVAPSQSLSITCTVSGFSLPSYGVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLTISKDNSKSQVFLKMNSLQTDDTAIYYCARGGSDYDGFAYWGQGTLVTVS(配列番号28)-
single chain:GGGGSGGGGSGGGGS(配列番号29)-
mVkappa:DIVMTQSPSSLAVSAGEKVTMNCRSSQSLLSSRTRKNYLAWYQQKPGQSPKLLIYWASIRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLRTFGGGTKLEIK(配列番号30)-
hCkappa:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGECGAP(配列番号31)-
hCD28 transmembrane:TRFWVLVVVGGVLACYSLLVTVAFIIFWVR(配列番号32)-
hCD3 zeta:LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(配列番号34)-
hGITR intracellular domain:RSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV*(配列番号36)
28z GITRL CARについては、28z CAR発現プラスミドの配列番号25の塩基配列の3´側に隣接して、P2A-GITRL塩基配列:GGATCCGGCGCCACAAATTTTAGCCTCTTGAAGCAAGCCGGCGACGTGGAAGAGAATCCTGGGCCC(P2A塩基配列:配列番号37)-ATGACCCTGCACCCCAGCCCCATCACCTGCGAGTTCCTGTTCAGCACCGCCCTGATCAGCCCCAAGATGTGCCTGAGCCACCTGGAGAACATGCCCCTGAGCCACAGCAGAACCCAGGGCGCCCAGAGAAGCAGCTGGAAGCTGTGGCTGTTCTGCAGCATCGTGATGCTGCTGTTCCTGTGCAGCTTCAGCTGGCTGATCTTCATCTTCCTGCAGCTGGAGACCGCCAAGGAGCCCTGCATGGCCAAGTTCGGCCCCCTGCCCAGCAAGTGGCAGATGGCCAGCAGCGAGCCCCCCTGCGTGAACAAGGTGAGCGACTGGAAGCTGGAGATCCTGCAGAACGGCCTGTACCTGATCTACGGCCAGGTGGCCCCCAACGCCAACTACAACGACGTGGCCCCCTTCGAGGTGAGACTGTACAAGAACAAGGACATGATCCAGACCCTGACCAACAAGAGCAAGATCCAGAACGTGGGCGGCACCTACGAGCTGCACGTGGGCGACACCATCGACCTGATCTTCAACAGCGAGCACCAGGTGCTGAAGAACAACACCTACTGGGGCATCATCCTGCTGGCCAACCCCCAGTTCATCAGC(GITRL塩基配列:配列番号38)が組み込まれてなる発現プラスミドを、人工遺伝子及びPCRを利用して、作製した。なお、P2A-GITRLアミノ酸配列は次の通りである:GSGATNFSLLKQAGDVEENPGP(P2Aアミノ酸配列:配列番号39)-MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS(GITRLアミノ酸配列:配列番号40)。
上記で構築したプラスミドDNAをPlat-A細胞に導入してレトロウィルスを作製し、それを培養ヒトPBMCに感染させてCAR導入T細胞を得て、フローサイトメトリーでCAR発現を調べた。図6~11に示すように alpha/beta T細胞、図12~13に示すように gamma/delta T細胞におけるCAR及びリガンドの発現が確認できた。これがエフェクター細胞である。また、CARの発現効率及び発現強度(mean fluorescent intensity, MFIで示す)はGITRLの共発現により増強したことが確認された(図6~8)。
CAR T細胞は、標的であるAS細胞と共培養すると活性化し、IFNg, TNFaを発現する。また、CD107aが細胞表面に移行する。これらの反応は、多機能性の反応が起きたことを示している。今回作製したCAR T細胞はいずれもAS細胞との共培養によって活性化し、これらの反応が起きたことが確認された(図14~16)。
AS細胞に対するエフェクター細胞の細胞傷害作用を、xCelligenceによる継時的変化によって検討した。その結果、GD2 CARがalpha/beta T細胞、gamma/delta T細胞でも十分な細胞傷害活性を有することが示された(図17及び18)。このことは、非RI細胞傷害性試験でも認められた(図19)。
GD2陽性Kelly細胞20000個をE-plate上で20時間培養したのち、各エフェクター細胞(alpha/beta) 40000個を添加培養し、cell indexを継時的に追跡した。Cell indexはE-plate上のKelly細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のKelly細胞数を1として標準化した。グラフは平均値(n=2)を示す。 GD2 28z, GD2 zG, GITRL共発現GD2 28z CAR T細胞による効果的な細胞傷害が観察され、CAR導入の無いPBMCによる細胞傷害は観察されなかった(図20)。
GD2陰性のSK-N-SH細胞20000個をE-plateにて18時間培養したのち、そこにエフェクター細胞(alpha/beta) 40000個を作用させ、cell indexを継時的に追跡したCell indexはE-plate上のSK-N-SH細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のSK-N-SH細胞数を1として標準化した。グラフは平均値(n=2)を示す。 GD2 28z, GD2 zG, GITRL共発現GD2 28z CAR T細胞による細胞傷害は観察されなかった(図21)。
GD2陽性のHs578T-Luc細胞15000個をE-plateにて20時間培養したのち、そこにエフェクター細胞(alpha/beta) 40000個を作用させ、cell indexを継時的に追跡した。 Cell indexはE-plate上のHs578T-Luc細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のHs578T-Luc細胞数を1として標準化した。グラフは平均値(n=2)を示す。 GD2 28z, GD2 zG, GITRL共発現GD2 28z CAR T細胞による効果的な細胞傷害が観察され、CAR導入の無いPBMCによる細胞傷害は観察されなかった(図22)。
GD2陰性のBT549-Luc細胞20000個をE-plateにて18時間培養したのち、そこにエフェクター細胞(alpha/beta) 40000個を作用させ、cell indexを継時的に追跡した。 Cell indexはE-plate上のBT549-Luc細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のBT549-Luc細胞数を1として標準化した。グラフは平均値(n=2)を示す。 GD2 28z, GITRL共発現GD2 28z CAR T細胞による細胞傷害は観察されなかった(図23)。
GD2陽性のKelly細胞20000個をE-plateにて20時間培養したのち、そこにエフェクター細胞(alpha/beta) 30000個を作用させ、cell indexを継時的に追跡した。 1日後、GD2 28z, GITRL共発現GD2 28z CAR T細胞による効果的な細胞傷害が観察された。このエフェクター細胞を回収し、24時間E-plate上で培養したKelly細胞と連続的に共培養し、cell indexの変化を継時的に記録した。 Cell indexはE-plate上のKelly細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のKelly細胞数を1として標準化した。グラフは平均値(n=2)を示す。 連続した2回目の細胞傷害性試験で、GITRL共発現28z CAR-T細胞が28z CARに比較して、より強い細胞傷害活性を保持していた(図24)。
D8細胞はGD2陰性の小細胞肺がんSK-LC-17にGD3 synthaseとGD2 synthase遺伝子を導入し、G418選択によって確立したGD2陽性細胞株である。GD2陽性のD8細胞10000個をE-plateにて18時間培養したのち、そこにエフェクター細胞(alpha/beta) 30000個を作用させ、cell indexを継時的に追跡した。 Cell indexはE-plate上のD8細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のD8細胞数を1として標準化した。グラフは平均値(n=2)を示す。 GD2 28z, GD2 zG, GITRL共発現GD2 28z CAR T細胞による効果的な細胞傷害が観察され、CAR導入の無いPBMCによる細胞傷害は観察されなかった(図25)。
C2細胞はGD2陰性の小細胞肺がんSK-LC-17にpCDNA3.1neoプラスミドを導入し、G418選択によって確立したGD2陰性細胞株である。GD2陰性のC2細胞10000個をE-plateにて18時間培養したのち、そこにエフェクター細胞(alpha/beta) 30000個を作用させ、cell indexを継時的に追跡した。 Cell indexはE-plate上のC2細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のC2細胞数を1として標準化した。グラフは平均値(n=2)を示す。 GD2 28z, GD2 zG, GITRL共発現GD2 28z CAR T細胞、 CAR導入の無いPBMCによる細胞傷害は観察されなかった(図26)。
GD2陽性のNCI-N417細胞20000個をE-plateにて34時間培養したのち、そこにエフェクター細胞(alpha/beta) 60000個を作用させ、cell indexを継時的に追跡した。 Cell indexはE-plate上のNCI-N417細胞数を反映する。Normalized cell indexは、エフェクター細胞と共培養する直前のNCI-N417細胞数を1として標準化した。グラフは平均値(n=2)を示す。 GD2 28z, GD2 zG, GITRL共発現GD2 28z CAR T細胞による効果的な細胞傷害が観察され、CAR導入の無いPBMCによる細胞傷害は観察されなかった(図27)。
Claims (13)
- 配列番号1で示されるアミノ酸配列を含む重鎖CDR1、配列番号2で示されるアミノ酸配列を含む重鎖CDR2、及び配列番号3で示されるアミノ酸配列を含む重鎖CDR3を含む重鎖可変領域、並びに/或いは
配列番号9で示されるアミノ酸配列を含む軽鎖CDR1、配列番号10で示されるアミノ酸配列を含む軽鎖CDR2、及び配列番号11で示されるアミノ酸配列を含む軽鎖CDR3を含む軽鎖可変領域
を含む、GD2結合性分子。 - 前記重鎖可変領域及び前記軽鎖可変領域を含む、請求項1に記載のGD2結合性分子。
- ガングリオシドGD1a、ガングリオシドGD1b、ガングリオシドGD3、ガングリオシドGM1、ガングリオシドGM3、ガングリオシドGT1b、及びラクトシルセラミドに対する結合性が、ガングリオシドGD2に対する結合性の1/2以下である、請求項1又は2に記載のGD2結合性分子。
- キメラ抗原受容体である、請求項1~3のいずれかに記載のGD2結合性分子。
- 前記重鎖可変領域及び前記軽鎖可変領域を含むscFvドメイン、膜貫通ドメイン、並びにTCRの細胞内ドメインを含むコア領域を含む、請求項4に記載のGD2結合性分子。
- 前記コア領域がさらに共刺激因子の細胞内ドメインを含む、請求項5に記載のGD2結合性分子。
- 前記コア領域のC末端側に、自己切断ペプチドドメインを介してGITRLドメインを含む、請求項5又は6に記載のGD2結合性分子。
- 抗体である、請求項1~3のいずれかに記載のGD2結合性分子。
- 請求項1~8のいずれかに記載のGD2結合性分子をコードする、ポリヌクレオチド。
- 請求項9に記載のポリヌクレオチドを含有する、細胞。
- 請求項4~7のいずれかに記載のGD2結合性分子をコードするポリヌクレオチドを含有する、キメラ抗原受容体T細胞又はキメラ抗原受容体NK細胞。
- 請求項11に記載のキメラ抗原受容体T細胞若しくはキメラ抗原受容体NK細胞、又は請求項8に記載のGD2結合性分子を含有する、医薬組成物。
- がんの治療又は予防用である、請求項12に記載の医薬組成物。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP20845660.8A EP4008347A4 (en) | 2019-08-01 | 2020-07-31 | GD2-BINDING MOLECULE |
US17/631,745 US20220275104A1 (en) | 2019-08-01 | 2020-07-31 | Gd2 binding molecule |
JP2021535464A JP7505784B2 (ja) | 2019-08-01 | 2020-07-31 | Gd2結合性分子 |
BR112022001649A BR112022001649A2 (pt) | 2019-08-01 | 2020-07-31 | Molécula de ligação ao gd2 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019142358 | 2019-08-01 | ||
JP2019-142358 | 2019-08-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021020564A1 true WO2021020564A1 (ja) | 2021-02-04 |
Family
ID=74228907
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2020/029446 WO2021020564A1 (ja) | 2019-08-01 | 2020-07-31 | Gd2結合性分子 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20220275104A1 (ja) |
EP (1) | EP4008347A4 (ja) |
JP (1) | JP7505784B2 (ja) |
BR (1) | BR112022001649A2 (ja) |
WO (1) | WO2021020564A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4008404A4 (en) * | 2019-08-01 | 2023-03-01 | Mie University | ANTIGEN RECEPTOR |
WO2023081485A1 (en) | 2021-11-08 | 2023-05-11 | Pacific Biosciences Of California, Inc. | Stepwise sequencing of a polynucleotide with a homogenous reaction mixture |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011526785A (ja) * | 2008-06-30 | 2011-10-20 | モーフオテク・インコーポレーテツド | 抗gd2抗体並びにそれに関連する方法及び使用 |
WO2012033885A1 (en) * | 2010-09-08 | 2012-03-15 | Baylor College Of Medicine | Immunotherapy of cancer using genetically engineered gd2-specific t cells |
JP2013532968A (ja) * | 2010-06-19 | 2013-08-22 | メモリアル スローン−ケタリング キャンサー センター | 抗gd2抗体 |
JP2015521607A (ja) * | 2012-06-18 | 2015-07-30 | アペイロン・バイオロジックス・アクチェンゲゼルシャフトAPEIRON Biologics AG | Gd2陽性癌を治療するための方法 |
WO2018023025A1 (en) * | 2016-07-28 | 2018-02-01 | Novartis Ag | Combination therapies of chimeric antigen receptors adn pd-1 inhibitors |
WO2018183888A2 (en) * | 2017-03-31 | 2018-10-04 | The Board Of Trustees Of The Leland Stanford Junior University | Methods of treating t cell exhaustion by inhibiting or modulating t cell receptor signaling |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201403972D0 (en) * | 2014-03-06 | 2014-04-23 | Ucl Business Plc | Chimeric antigen receptor |
WO2016134284A1 (en) * | 2015-02-19 | 2016-08-25 | University Of Florida Research Foundation, Inc. | Chimeric antigen receptors and uses thereof |
SG10202109108UA (en) * | 2017-06-21 | 2021-09-29 | Icell Gene Therapeutics Llc | Chimeric antigen receptors (cars), compositions and methods thereof |
IT201800007601A1 (it) * | 2018-07-30 | 2020-01-30 | Rigenerand Srl | Metodo per la produzione di cellule bi-funzionali per il trattamento di tumori |
-
2020
- 2020-07-31 JP JP2021535464A patent/JP7505784B2/ja active Active
- 2020-07-31 US US17/631,745 patent/US20220275104A1/en active Pending
- 2020-07-31 EP EP20845660.8A patent/EP4008347A4/en active Pending
- 2020-07-31 BR BR112022001649A patent/BR112022001649A2/pt unknown
- 2020-07-31 WO PCT/JP2020/029446 patent/WO2021020564A1/ja unknown
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011526785A (ja) * | 2008-06-30 | 2011-10-20 | モーフオテク・インコーポレーテツド | 抗gd2抗体並びにそれに関連する方法及び使用 |
JP2013532968A (ja) * | 2010-06-19 | 2013-08-22 | メモリアル スローン−ケタリング キャンサー センター | 抗gd2抗体 |
WO2012033885A1 (en) * | 2010-09-08 | 2012-03-15 | Baylor College Of Medicine | Immunotherapy of cancer using genetically engineered gd2-specific t cells |
JP2015521607A (ja) * | 2012-06-18 | 2015-07-30 | アペイロン・バイオロジックス・アクチェンゲゼルシャフトAPEIRON Biologics AG | Gd2陽性癌を治療するための方法 |
WO2018023025A1 (en) * | 2016-07-28 | 2018-02-01 | Novartis Ag | Combination therapies of chimeric antigen receptors adn pd-1 inhibitors |
WO2018183888A2 (en) * | 2017-03-31 | 2018-10-04 | The Board Of Trustees Of The Leland Stanford Junior University | Methods of treating t cell exhaustion by inhibiting or modulating t cell receptor signaling |
Non-Patent Citations (4)
Title |
---|
FUKUDA M; HORIBE K; FURUKAWA K: "Enhancement of in vitro and in vivo anti-tumor activity of anti-GD2 monoclonal antibody 220-51 against human neuroblastoma by granulocyte-macrophage colony-stimulating factor and granulocyte colony- stimulating factor", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, vol. 2, no. 4, 1 October 1998 (1998-10-01), pages 471 - 475, XP009526266, ISSN: 1107-3756, DOI: 10.3892/ijmm.2.4.471 * |
KARLIN SALTSCHUL SF.: "Applications and statistics for multiple high-scoring segments in molecular sequences", PROC NATL ACAD SCI USA., vol. 90, 1993, pages 5873 - 7, XP001030852, DOI: 10.1073/pnas.90.12.5873 |
KARLIN SALTSCHUL SF: "Methods for assessing the statistical significance of molecular sequence features by using general scorings schemes", PROC NATL ACAD SCI USA., vol. 87, 1990, pages 2264 - 2268 |
See also references of EP4008347A4 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4008404A4 (en) * | 2019-08-01 | 2023-03-01 | Mie University | ANTIGEN RECEPTOR |
WO2023081485A1 (en) | 2021-11-08 | 2023-05-11 | Pacific Biosciences Of California, Inc. | Stepwise sequencing of a polynucleotide with a homogenous reaction mixture |
Also Published As
Publication number | Publication date |
---|---|
EP4008347A4 (en) | 2023-03-08 |
EP4008347A1 (en) | 2022-06-08 |
BR112022001649A2 (pt) | 2022-03-22 |
JP7505784B2 (ja) | 2024-06-25 |
JPWO2021020564A1 (ja) | 2021-02-04 |
US20220275104A1 (en) | 2022-09-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6571527B2 (ja) | 二重特異性抗体 | |
EP3733715A1 (en) | Triabody, preparation method and use thereof | |
KR20240156612A (ko) | 항b7-h3의 모노클로널 항체 및 그가 세포 치료 중에서의 응용 | |
JP2022515487A (ja) | クローディン18.2結合部分およびその利用 | |
TW202045545A (zh) | 白蛋白結合抗體及其用途 | |
CN104684928A (zh) | 通过半胱氨酸突变和μ尾端多聚化的抗体或融合蛋白 | |
US20200024358A1 (en) | Trispecific antigen binding proteins | |
JP2023178323A (ja) | メソテリン及びcd137結合分子 | |
EP4257610A1 (en) | Ror1-targeting antibody and use thereof | |
CA3056374A1 (en) | Anti-psma antibodies and use thereof | |
US20220144960A1 (en) | Cd30-binding moieties, chimeric antigen receptors, and uses thereof | |
WO2022026759A1 (en) | T cells and chimeric stimulating receptors and uses thereof | |
US20220185882A1 (en) | Artificial immunosurveillance chimeric antigen receptor (ai-car) and cells expressing the same | |
CN115052901A (zh) | 靶向bcma的单结构域抗体和嵌合抗原受体及其使用方法 | |
JP7505784B2 (ja) | Gd2結合性分子 | |
CN114981305A (zh) | Claudin18.2结合部分及其用途 | |
WO2022224997A1 (ja) | 抗cldn4-抗cd137二重特異性抗体 | |
US20230372484A1 (en) | Chimeric antigen receptors for treatment of cancer | |
EP4242309A1 (en) | Chimeric antigen receptor | |
CA3226700A1 (en) | Agents encoding cldn6 and cds binding elements for treating cldn6-positive cancers | |
IL305181A (en) | Cell therapy compositions and methods for modulating TGF-B signaling | |
JP2020506716A (ja) | Cd43の固有のシアログリコシル化がん関連エピトープを標的化するモノクローナル抗体 | |
WO2022116079A1 (zh) | 一种抗ceacam5的人源化抗体及其制备方法和用途 | |
WO2022124282A1 (ja) | Prame結合性分子 | |
RU2824670C2 (ru) | Gd2-связывающая молекула |
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: 20845660 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2021535464 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112022001649 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 2020845660 Country of ref document: EP Effective date: 20220301 |
|
ENP | Entry into the national phase |
Ref document number: 112022001649 Country of ref document: BR Kind code of ref document: A2 Effective date: 20220128 |