JP2020103280A - Method for producing antibody having improved antibody-dependent cytotoxic activity - Google Patents
Method for producing antibody having improved antibody-dependent cytotoxic activity Download PDFInfo
- Publication number
- JP2020103280A JP2020103280A JP2019224246A JP2019224246A JP2020103280A JP 2020103280 A JP2020103280 A JP 2020103280A JP 2019224246 A JP2019224246 A JP 2019224246A JP 2019224246 A JP2019224246 A JP 2019224246A JP 2020103280 A JP2020103280 A JP 2020103280A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- added
- cells
- human
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 32
- 230000001472 cytotoxic effect Effects 0.000 title abstract description 3
- 230000001419 dependent effect Effects 0.000 title abstract 2
- 229910001437 manganese ion Inorganic materials 0.000 claims abstract description 82
- 210000004962 mammalian cell Anatomy 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 31
- 238000012258 culturing Methods 0.000 claims abstract description 21
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims description 68
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims description 40
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 19
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 239000012533 medium component Substances 0.000 claims description 7
- 238000012544 monitoring process Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 92
- 239000002609 medium Substances 0.000 description 66
- 230000000694 effects Effects 0.000 description 38
- 108090000623 proteins and genes Proteins 0.000 description 29
- 239000013612 plasmid Substances 0.000 description 27
- 108091008146 restriction endonucleases Proteins 0.000 description 24
- 238000004458 analytical method Methods 0.000 description 21
- 230000000052 comparative effect Effects 0.000 description 20
- 239000000243 solution Substances 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 19
- 230000012010 growth Effects 0.000 description 16
- 239000000047 product Substances 0.000 description 14
- 230000007704 transition Effects 0.000 description 13
- 241000588724 Escherichia coli Species 0.000 description 12
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 12
- 150000001413 amino acids Chemical group 0.000 description 11
- 108091033319 polynucleotide Proteins 0.000 description 11
- 239000002157 polynucleotide Substances 0.000 description 11
- 102000040430 polynucleotide Human genes 0.000 description 11
- 101150074155 DHFR gene Proteins 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 8
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 8
- 229930027917 kanamycin Natural products 0.000 description 8
- 229960000318 kanamycin Drugs 0.000 description 8
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 8
- 229930182823 kanamycin A Natural products 0.000 description 8
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 7
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 7
- 102000023732 binding proteins Human genes 0.000 description 7
- 108091008324 binding proteins Proteins 0.000 description 7
- 229910002091 carbon monoxide Inorganic materials 0.000 description 7
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 7
- 229960000485 methotrexate Drugs 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 230000003248 secreting effect Effects 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 229940099596 manganese sulfate Drugs 0.000 description 5
- 235000007079 manganese sulphate Nutrition 0.000 description 5
- 239000011702 manganese sulphate Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 230000004544 DNA amplification Effects 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 239000007979 citrate buffer Substances 0.000 description 4
- 230000037433 frameshift Effects 0.000 description 4
- SXTAYKAGBXMACB-UHFFFAOYSA-N methionine sulfoximine Chemical compound CS(=N)(=O)CCC(N)C(O)=O SXTAYKAGBXMACB-UHFFFAOYSA-N 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 108010022394 Threonine synthase Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940125644 antibody drug Drugs 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000009827 complement-dependent cellular cytotoxicity Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 102000004419 dihydrofolate reductase Human genes 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 150000002697 manganese compounds Chemical class 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- QRBLKGHRWFGINE-UGWAGOLRSA-N 2-[2-[2-[[2-[[4-[[2-[[6-amino-2-[3-amino-1-[(2,3-diamino-3-oxopropyl)amino]-3-oxopropyl]-5-methylpyrimidine-4-carbonyl]amino]-3-[(2r,3s,4s,5s,6s)-3-[(2s,3r,4r,5s)-4-carbamoyl-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)- Chemical compound N=1C(C=2SC=C(N=2)C(N)=O)CSC=1CCNC(=O)C(C(C)=O)NC(=O)C(C)C(O)C(C)NC(=O)C(C(O[C@H]1[C@@]([C@@H](O)[C@H](O)[C@H](CO)O1)(C)O[C@H]1[C@@H]([C@](O)([C@@H](O)C(CO)O1)C(N)=O)O)C=1NC=NC=1)NC(=O)C1=NC(C(CC(N)=O)NCC(N)C(N)=O)=NC(N)=C1C QRBLKGHRWFGINE-UGWAGOLRSA-N 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- LTQCLFMNABRKSH-UHFFFAOYSA-N Phleomycin Natural products N=1C(C=2SC=C(N=2)C(N)=O)CSC=1CCNC(=O)C(C(O)C)NC(=O)C(C)C(O)C(C)NC(=O)C(C(OC1C(C(O)C(O)C(CO)O1)OC1C(C(OC(N)=O)C(O)C(CO)O1)O)C=1NC=NC=1)NC(=O)C1=NC(C(CC(N)=O)NCC(N)C(N)=O)=NC(N)=C1C LTQCLFMNABRKSH-UHFFFAOYSA-N 0.000 description 2
- 108010035235 Phleomycins Proteins 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- 108010084455 Zeocin Proteins 0.000 description 2
- 229930189065 blasticidin Natural products 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 2
- 102000005396 glutamine synthetase Human genes 0.000 description 2
- 108020002326 glutamine synthetase Proteins 0.000 description 2
- 229940027941 immunoglobulin g Drugs 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 229940100601 interleukin-6 Drugs 0.000 description 2
- 108040006858 interleukin-6 receptor activity proteins Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 108700026220 vif Genes Proteins 0.000 description 2
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 101150074355 GS gene Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010058683 Immobilized Proteins Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 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
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- -1 glutamine Chemical class 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000055277 human IL2 Human genes 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 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
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
本発明は、高い抗体依存性細胞傷害(ADCC)活性を有した抗体を製造する方法に関する。より詳しくは、抗体を発現可能な宿主を培養する工程を最適化することで、当該宿主が発現する抗体が有するADCC作用を向上させる方法に関する。 The present invention relates to a method for producing an antibody having high antibody-dependent cellular cytotoxicity (ADCC) activity. More specifically, it relates to a method for improving the ADCC action of an antibody expressed by the host by optimizing the step of culturing a host capable of expressing the antibody.
現在、組換えタンパク質は幅広い分野で使用されている。近年の抗体医薬品に代表されるバイオ医薬品の成長によりその重要性はさらに高まっている。組換えタンパク質は主に大腸菌、酵母、昆虫細胞、哺乳細胞を宿主として製造されているが、発現させた組換えタンパク質の立体構造や、糖鎖付加といった翻訳後修飾等の理由から哺乳動物細胞を宿主として用いた組換えタンパク質発現系の重要性が増している。特にチャイニーズハムスター卵巣細胞(以下、CHO細胞)は多くの組換えタンパク質発現の宿主として用いられる。また組換えCHO細胞由来の組換えタンパク質は、医薬品として使用できる安全性が確認されていることから、抗体医薬品を製造する際の宿主として最もよく使用される哺乳動物細胞である。 Currently, recombinant proteins are used in a wide range of fields. With the recent growth of biopharmaceuticals represented by antibody pharmaceuticals, their importance is increasing. Recombinant proteins are mainly produced in Escherichia coli, yeast, insect cells, and mammalian cells as hosts. However, because of the three-dimensional structure of the expressed recombinant protein and post-translational modification such as glycosylation, The importance of recombinant protein expression systems used as hosts is increasing. In particular, Chinese hamster ovary cells (hereinafter referred to as CHO cells) are used as a host for expressing many recombinant proteins. In addition, since the recombinant protein derived from recombinant CHO cells has been confirmed to be safe to use as a drug, it is a mammalian cell most often used as a host when producing an antibody drug.
抗体医薬品はモノクローナル抗体を主成分とした医薬品であるが、その治療効果には信号伝達阻害、細胞死(Apoptosis)の誘導、抗体依存性細胞傷害(ADCC)作用や補体依存性細胞傷害(CDC)作用などがある。このうちADCC活性やCDC活性はエフェクター機能と呼ばれており免疫メカニズムを誘導することで癌等の目的細胞を傷害するため重要である。特にADCC活性は抗体医薬品の細胞傷害作用には重要な活性である。 The antibody drug is a drug whose main component is a monoclonal antibody, and its therapeutic effects include signal transduction inhibition, induction of cell death (Apoptosis), antibody-dependent cellular cytotoxicity (ADCC) action and complement-dependent cellular cytotoxicity (CDC). ) There is an action. Among these, ADCC activity and CDC activity are called effector functions, and are important because they induce an immune mechanism to damage target cells such as cancer. In particular, ADCC activity is an important activity for the cytotoxic action of antibody drugs.
抗体が有するADCC活性を向上させる方法として、抗体のFc領域にある特定のアミノ酸残基を他のアミノ酸残基に置換する方法と、抗体のFc領域に付加する糖鎖を改変する方法に大別される。抗体のFc領域に付加する糖鎖には、特定のアミノ酸配列(例えば、Asn−X−Ser/Thr)(Xは任意のアミノ酸残基を示す)のアスパラギン(Asn)基の側鎖に付加するN型糖鎖と、セリン(Ser)またはスレオニン(Thr)残基の側鎖に結合するO型糖鎖がある。このうちN型糖鎖のコアフコースを欠損させることでADCC活性が向上する報告がされている(非特許文献1)。 Methods for improving the ADCC activity of an antibody are roughly classified into a method of substituting a specific amino acid residue in the Fc region of the antibody with another amino acid residue and a method of modifying the sugar chain added to the Fc region of the antibody. To be done. The sugar chain added to the Fc region of the antibody is added to the side chain of the asparagine (Asn) group of a specific amino acid sequence (for example, Asn-X-Ser/Thr) (X represents an arbitrary amino acid residue). There are N-type sugar chains and O-type sugar chains that bind to the side chains of serine (Ser) or threonine (Thr) residues. Among them, it has been reported that the ADCC activity is improved by deleting the core fucose of the N-type sugar chain (Non-Patent Document 1).
抗体を発現可能な哺乳動物細胞を培養し、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収することで、前記抗体を製造する際、前記哺乳動物細胞の培養条件を変化させると、前記抗体のFc領域に付加するN型糖鎖の構造が変化することが知られている(特許文献1)。しかしながら、実際に抗体のFc領域へ付加する糖鎖は不均一であり、前記糖鎖の構造とADCC活性との関係を完全に結び付けることは難しい。従って、糖鎖構造を制御してADCC活性の高い抗体を製造する条件、特に前記抗体を発現可能な哺乳動物細胞を培養する条件の構築は困難であった。 Culturing mammalian cells capable of expressing the antibody, by recovering the antibody expressed by the mammalian cells contained in the obtained culture, when producing the antibody, the culture conditions of the mammalian cells It is known that the structure of the N-type sugar chain added to the Fc region of the antibody is changed by changing it (Patent Document 1). However, the sugar chains actually added to the Fc region of the antibody are heterogeneous, and it is difficult to completely link the relationship between the sugar chain structure and ADCC activity. Therefore, it was difficult to construct conditions for producing an antibody with high ADCC activity by controlling the sugar chain structure, particularly for culturing mammalian cells capable of expressing the antibody.
本発明の課題は、抗体を発現可能な哺乳動物細胞を培養する工程と、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収する工程とを含む、抗体の製造方法において、高い抗体依存性細胞傷害活性を有する抗体を製造する方法を提供することにある。 The subject of the present invention comprises a step of culturing a mammalian cell capable of expressing an antibody, and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture, in a method for producing an antibody Another object of the present invention is to provide a method for producing an antibody having high antibody-dependent cellular cytotoxicity.
本発明者らは上記の課題を解決すべく鋭意検討した結果、抗体を発現可能な哺乳動物細胞を培養する工程において、前記哺乳動物細胞を一定量のマンガンイオンを添加した培地で培養することで、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体の抗体依存性細胞傷害が向上することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that in the step of culturing a mammalian cell capable of expressing an antibody, the mammalian cell is cultured in a medium containing a fixed amount of manganese ion. The inventors have found that the antibody-dependent cellular cytotoxicity of the antibody expressed by the mammalian cells contained in the obtained culture is improved, and completed the present invention.
すなわち本発明は、以下に記載の態様を包含する。 That is, the present invention includes the embodiments described below.
(1)抗体を発現可能な哺乳動物細胞を培養する工程と得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収する工程とを含む抗体の製造方法において、前記培養工程を0.01μMから50μMのマンガンイオンを添加した培地で行なうことで、抗体依存性細胞傷害活性が向上した抗体を製造する方法。 (1) In a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering an antibody expressed by the mammalian cell contained in the obtained culture, the culturing step comprising: A method for producing an antibody having improved antibody-dependent cellular cytotoxicity, which is carried out in a medium supplemented with 0.01 μM to 50 μM manganese ion.
(2)抗体がヒトFc領域を含む抗体である、(1)に記載の製造方法。 (2) The production method according to (1), wherein the antibody comprises a human Fc region.
(3)(2)に記載の製造方法で得られた抗体とヒトFcγRIIIaとの親和性を評価することで、(2)に記載の製造方法における培養工程をモニタリングする方法。 (3) A method of monitoring the culture step in the production method according to (2) by evaluating the affinity between the antibody obtained by the production method according to (2) and human FcγRIIIa.
(4)(2)に記載の製造方法で得られた抗体とヒトFcγRIIIaとの親和性を評価することで、(2)に記載の製造方法における培地成分を評価する方法。 (4) A method for evaluating the medium components in the production method according to (2) by evaluating the affinity between the antibody obtained by the production method according to (2) and human FcγRIIIa.
(5)(2)に記載の製造方法で得られた抗体とヒトFcγRIIIaとの親和性評価を、(2)に記載の製造方法で得られた抗体とヒトFcγRIIIa固定化分離剤との結合力に基づき行なう、(3)または(4)に記載の方法。 (5) The affinity evaluation between the antibody obtained by the production method described in (2) and human FcγRIIIa is evaluated by the binding force between the antibody obtained by the production method described in (2) and the human FcγRIIIa-immobilized separating agent. The method according to (3) or (4), which is performed based on
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の製造方法は、抗体を発現可能な哺乳動物細胞を培養する際、0.01μMから50μMのマンガンイオンを培地に添加して培養することを特徴としている。培地中のマンガンイオンが前述した濃度範囲であれば、細胞の増殖性や抗体の生産性が著しく阻害されることはない。マンガンイオンの培地への添加は、水溶液中でマンガンイオンとして存在可能なマンガン化合物を添加すればよい。前記マンガン化合物の例として、塩化マンガン(MnCl2)や硫酸マンガン(MnSO4)が例示できる。マンガン化合物の添加量は、培地中のマンガンイオンが前述した濃度範囲となるよう添加すればよく、0.03μMから30μMとなるよう添加すると好ましく、0.1μMから10μMとなるよう添加するとより好ましく、1μMから10μMとなるよう添加するとさらにより好ましい。 The production method of the present invention is characterized by adding 0.01 to 50 μM manganese ion to a medium when culturing a mammalian cell capable of expressing an antibody. When the manganese ion concentration in the medium is within the above-mentioned concentration range, cell proliferation and antibody productivity are not significantly impaired. The manganese ion may be added to the medium by adding a manganese compound that can exist as manganese ion in the aqueous solution. Examples of the manganese compound include manganese chloride (MnCl 2 ) and manganese sulfate (MnSO 4 ). The amount of the manganese compound added may be such that the manganese ion in the medium is in the concentration range described above, preferably 0.03 μM to 30 μM, and more preferably 0.1 μM to 10 μM. It is even more preferable to add 1 μM to 10 μM.
本発明の製造方法で使用する哺乳動物細胞は、製造対象抗体を発現可能な細胞であれば特に制限はない。一例を示すと、CHO細胞(CHO−K1、CHO−S、CHO−DG44およびCHO−DXB11)、マウス骨髄腫由来細胞(SP2/0、NS0)、ヒト胎児腎臓由来細胞(HEK細胞)、ヒト白血病細胞由来細胞(HL−60細胞)、ヒト子宮頸癌由来細胞(HeLa細胞)およびアフリカミドリザルの腎細胞由来細胞(COS細胞)があげられる。中でも組換え抗体製造に汎用されるCHO細胞の使用が好ましい。 The mammalian cells used in the production method of the present invention are not particularly limited as long as they can express the antibody to be produced. As an example, CHO cells (CHO-K1, CHO-S, CHO-DG44 and CHO-DXB11), mouse myeloma-derived cells (SP2/0, NS0), human fetal kidney-derived cells (HEK cells), human leukemia Examples thereof include cell-derived cells (HL-60 cells), human cervical cancer-derived cells (HeLa cells) and African green monkey kidney cell-derived cells (COS cells). Above all, it is preferable to use CHO cells which are generally used for producing recombinant antibodies.
本発明の製造方法で使用する培地は、0.01μMから50μMのマンガンイオンを含み、かつ宿主である哺乳動物細胞が生育し抗体を発現可能な培地であれば、特に限定はない。一例を示すと、動物由来の血清が必要な培地(RPMI1640、D−MEM等)や化学的に成分が決定されている培地(BalanCD CHO Growth A[Irvine Scientific社]、FreeStyle CHO Expression MediumCD[Thermo Fisher社]、OptiCHO[Thermo Fisher社]、EX−CELL CD CHO Fusion/EX−CELL Advanced CHO Fed−batch Medium[Merck社]およびCHOgro[Mirus社])に前述した濃度のマンガンイオンを添加した培地があげられる。さらに前述した培地に、栄養素、ホルモン、成長因子、特定イオン(ナトリウム、カリウム、カルシウム、マグネシウム等)、ビタミン、ヌクレオシド、ヌクレオチド、グルタミンのようなアミノ酸、無機塩(銅、亜鉛、コバルト、ニッケル等)、脂質、グルコースをはじめとする培地構成成分が含まれていてもよい。またG418、ピューロマイシン、ブラストサイジン、ゼオシン、ハイグロマイシン、フレオマイシン、カナマイシン、アンピシリンなどの抗生物質をさらに添加してもよい。 The medium used in the production method of the present invention is not particularly limited as long as it contains 0.01 μM to 50 μM manganese ion and can grow mammalian cells as a host and express antibodies. As an example, a medium that requires animal-derived serum (RPMI1640, D-MEM, etc.) or a medium whose components have been chemically determined (BalanCD CHO Growth A [Irvine Scientific], FreeStyle CHO Expression MediumCD[ThermoFertherFermo ], OptiCHO [Thermo Fisher], EX-CELL CD CHO Fusion/EX-CELL Advanced CHO Fed-batch Medium [Merck], and CHOgro [Mirus] with the above-mentioned concentration of the manganese ion added medium. To be Furthermore, nutrients, hormones, growth factors, specific ions (sodium, potassium, calcium, magnesium, etc.), vitamins, nucleosides, nucleotides, amino acids such as glutamine, inorganic salts (copper, zinc, cobalt, nickel, etc.) can be added to the aforementioned medium. , Mediums such as lipids and glucose may be contained. Further, antibiotics such as G418, puromycin, blasticidin, zeocin, hygromycin, phleomycin, kanamycin and ampicillin may be further added.
本発明の製造方法における培養工程は、宿主として用いる哺乳動物細胞や前記細胞で発現させる抗体に応じて適宜行なえばよい。一例として、
前述した濃度のマンガンイオンを添加した培地を入れたフラスコに、抗体を発現可能な哺乳動物細胞を接種後、当該フラスコを振盪させて培養してもよく、
前述した濃度のマンガンイオンを添加した培地を入れたバイオリアクターに、抗体を発現可能な哺乳動物細胞を接種後、回分培養、半回分培養(流加培養ともいう)、潅流培養またはそれらの組合せにより培養してもよい。
哺乳動物細胞がCHO細胞の場合、5%から8%のCO2存在下、温度30℃から37℃
、pH6.8から7.4で培養することが好ましい。
The culturing step in the production method of the present invention may be appropriately performed depending on the mammalian cell used as a host and the antibody expressed by the cell. As an example,
A flask containing a medium containing the above-mentioned concentration of manganese ion is inoculated with mammalian cells capable of expressing the antibody, and the flask may be shaken to be cultured.
After inoculating mammalian cells capable of expressing antibody into a bioreactor containing a medium to which manganese ions are added at the above-mentioned concentration, batch culture, semi-batch culture (also referred to as fed-batch culture), perfusion culture, or a combination thereof is performed. You may culture.
When the mammalian cells are CHO cells, the temperature is 30°C to 37°C in the presence of 5% to 8% CO 2.
It is preferable to culture at pH 6.8 to 7.4.
本発明の方法で製造する抗体の一例として、ヒトFc領域を含む抗体があげられる。具体的には、ヒト抗体、ヒト化抗体、ヒトと他の動物(マウスなど)とのキメラ抗体、ヒトFc融合タンパク質などがあげられる。ヒトFc領域を含む抗体がイムノグロブリンG(IgG)の場合、4つのサブクラス(IgG1、IgG2、IgG3、IgG4)が知られているが、このうちIgG1とIgG3は抗体依存性細胞傷害(ADCC)活性が高い点で、本発明の方法で製造する抗体の好ましい態様といえる。さらにIgG1はADCC活性が特に高く、本発明の方法で製造する抗体の特に好ましい態様といえる。 An example of the antibody produced by the method of the present invention is an antibody containing a human Fc region. Specific examples thereof include human antibodies, humanized antibodies, chimeric antibodies of humans and other animals (mouse, etc.), human Fc fusion proteins and the like. When the antibody containing the human Fc region is immunoglobulin G (IgG), four subclasses (IgG1, IgG2, IgG3, IgG4) are known. Of these, IgG1 and IgG3 have antibody-dependent cellular cytotoxicity (ADCC) activity. It can be said that it is a preferred embodiment of the antibody produced by the method of the present invention because of its high value. Furthermore, IgG1 has a particularly high ADCC activity, which can be said to be a particularly preferred embodiment of the antibody produced by the method of the present invention.
本発明の製造方法で使用する、抗体を発現可能な哺乳動物細胞は、前記抗体をコードするポリヌクレオチドを含む発現ベクターで、前記哺乳動物細胞を形質転換し、作製すればよい。前記発現ベクターには、プロモーターおよび前記抗体をコードするポリヌクレオチドの他に、ポリAや、組換え抗体の分泌発現に必要な分泌シグナルや、遺伝子増幅マーカー遺伝子や、宿主選択に用いる抗生物質耐性遺伝子や、遺伝子組換えのために用いる哺乳動物細胞以外の宿主での複製開始点等をさらに含んでもよい。 The mammalian cell capable of expressing an antibody used in the production method of the present invention may be produced by transforming the mammalian cell with an expression vector containing a polynucleotide encoding the antibody. In the expression vector, in addition to the promoter and the polynucleotide encoding the antibody, polyA, a secretory signal required for secretory expression of a recombinant antibody, a gene amplification marker gene, and an antibiotic resistance gene used for host selection Alternatively, a replication origin in a host other than mammalian cells used for gene recombination may be further included.
前記ポリAはターミネーションシグナルを含んでいれば特に制限はなく、一例として、発現させる抗体由来のポリA、SV40ウイルスゲノム由来のポリA、ヘルペスウイルスチミジンキナーゼのポリA、ウシ成長ホルモン由来のポリA、ウサギのβーグロビン遺伝子由来のポリAがあげられる。 The poly A is not particularly limited as long as it contains a termination signal, and examples thereof include poly A derived from an antibody to be expressed, poly A derived from the SV40 virus genome, poly A of herpesvirus thymidine kinase, and poly A derived from bovine growth hormone. , Poly A derived from the rabbit β-globin gene.
前記分泌シグナルは発現抗体を分泌すれば特に制限はなく、その一例としては、発現させる組換え抗体由来の分泌シグナル、ヒトインターロイキン2(ILー2)の分泌シグナル、アズロシジン前駆体の分泌シグナル、ヒト血清アルブミンの分泌シグナルがあげられる。 The secretory signal is not particularly limited as long as it secretes the expressed antibody, and examples thereof include a secretory signal derived from a recombinant antibody to be expressed, a secretory signal of human interleukin 2 (IL-2), a secretory signal of an azulocidin precursor, An example is a human serum albumin secretion signal.
前記遺伝子増幅マーカー遺伝子は、遺伝子増幅させる方法に適した遺伝子を用いればよい。例えばジヒドロ葉酸レダクターゼ(dhfr)/メトトレキサート(MTX)法を用いる場合はdhfr遺伝子を、グルタミン合成酵素(GS)/メチオニンスルホキシミン(MSX)法を用いる場合はGS遺伝子を、それぞれ用いればよい。 As the gene amplification marker gene, a gene suitable for the method of gene amplification may be used. For example, the dhfr gene may be used when the dihydrofolate reductase (dhfr)/methotrexate (MTX) method is used, and the GS gene may be used when the glutamine synthetase (GS)/methionine sulfoximine (MSX) method is used.
前記抗生物質耐性遺伝子は、宿主選択に用いる抗生物質に対応した耐性遺伝子を選択すればよく、一例として、G418耐性遺伝子、ピューロマイシン耐性遺伝子、ブラストサイジン耐性遺伝子、ゼオシン耐性遺伝子、ハイグロマイシン耐性遺伝子、フレオマイシン耐性遺伝子があげられる。 As the antibiotic resistance gene, a resistance gene corresponding to an antibiotic used for host selection may be selected, and as an example, G418 resistance gene, puromycin resistance gene, blasticidin resistance gene, zeocin resistance gene, hygromycin resistance gene. , And the phleomycin resistance gene.
前記複製開始点は、哺乳動物細胞以外の宿主が大腸菌である場合、大腸菌内でのコピー数が高くプラスミドDNAの収量が多い、ColE1が例示できる。 The replication origin can be exemplified by ColE1 which has a high copy number in E. coli and a high yield of plasmid DNA when the host other than mammalian cells is E. coli.
さらに前記発現ベクターには、プロモーターの働きを強めるためのエンハンサーをさらに含んでもよい。使用するエンハンサーに特に制限はなく、発現させる抗体や哺乳動物細胞を考慮し、適宜選択すればよい。一例としてサイトメガロウイルス(CMV)由来のエンハンサーがあげられる。 Furthermore, the expression vector may further include an enhancer for enhancing the function of the promoter. The enhancer used is not particularly limited and may be appropriately selected in consideration of the antibody to be expressed and mammalian cells. An example is a cytomegalovirus (CMV)-derived enhancer.
また哺乳動物に導入した遺伝子(抗体をコードするポリヌクレオチド)が発現しやすくするために、前記発現ベクターにLoxP遺伝子をさらに含ませてもよい。ゲノム中にLoxP遺伝子を含んだ宿主細胞へ発現ベクターを導入する際に、Creリコンビナーゼによる相同組換えを行なうことで宿主細胞のゲノムへ部位特異的に組換えタンパク質をコードするポリヌクレオチドを導入することができる。また、宿主細胞のゲノムへ部位特異的に組換えタンパク質をコードするポリヌクレオチドを導入する方法としてCRISPR/Cas9などを用いることもできる。 The expression vector may further contain a LoxP gene to facilitate expression of a gene (polynucleotide encoding an antibody) introduced into a mammal. When introducing an expression vector into a host cell containing the LoxP gene in its genome, homologous recombination with Cre recombinase is carried out to introduce a polynucleotide encoding a recombinant protein site-specifically into the genome of the host cell. You can In addition, CRISPR/Cas9 or the like can be used as a method for site-specifically introducing a polynucleotide encoding a recombinant protein into the genome of a host cell.
前記発現ベクターで哺乳動物細胞を形質転換するには、エレクトロポレーションやカチオニックリポソームを用いたリポフェクションなど、当業者が通常用いる形質転換法の中から、宿主として使用する哺乳動物細胞に合わせて適宜選択すればよい。 To transform a mammalian cell with the expression vector, electroporation, lipofection using a cationic liposome, or other transformation method commonly used by those skilled in the art is selected according to the mammalian cell used as the host. Just select it.
前述した方法で抗体を発現可能な哺乳動物細胞を培養後、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収することで、ADCC活性が向上した抗体を製造する。抗体の回収方法の一例として、前記得られた培養物から、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、ゲル濾過クロマトグラフィーなどのクロマトグラフィーによる精製操作を単独または組み合わせて抗体を回収する方法があげられる。前記方法は抗体を高効率かつ高純度に回収できる点で好ましい。 After culturing the mammalian cells capable of expressing the antibody by the method described above, the antibody expressed by the mammalian cells contained in the obtained culture is recovered to produce an antibody with improved ADCC activity. As an example of a method for recovering an antibody, a method for recovering an antibody from the obtained culture by a single or combination of purification operations by chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography and gel filtration chromatography. Can be given. The above method is preferable because the antibody can be recovered with high efficiency and high purity.
本発明で製造する抗体が、ヒトFc領域を含む抗体である場合、前記抗体とヒトFcγRIIIaとの親和性を評価することで、抗体を発現可能な哺乳動物細胞の培養状態(培養工程)をモニタリングできる。ヒトFc領域を含む抗体に付加するN型糖鎖を欠損させると前記抗体とヒトFcγRIIIaとの親和性が著しく低下する。またヒトFc領域を含む抗体が有するADCC活性は、当該Fc領域と免疫細胞表面上のヒトFcγRIIIaとの親和性(結合性)と関連することが知られている(Nordstrom.J,L, et al.,Breast Cancer Res.,13,6,(2011))。従って、ヒトFc領域を含む抗体とヒトFcγRIIIaとの親和性を評価することで、製造する抗体が有するADCC活性をモニタリングでき、抗体を発現可能な哺乳細胞の培養工程をモニタリングできる。一例として、得られたヒトFc領域を含む抗体とヒトFcγRIIIaとの親和性が低下した場合、当該抗体が有するADCC活性が低下しているため、当該抗体が有するADCC活性を向上すべく、マンガンイオン濃度などの培地成分や培養条件(二酸化炭素濃度、温度、pH、時間など)を適宜調整する。 When the antibody produced in the present invention is an antibody containing a human Fc region, the culture state (culture step) of mammalian cells capable of expressing the antibody is monitored by evaluating the affinity between the antibody and human FcγRIIIa. it can. When the N-type sugar chain added to the antibody containing the human Fc region is deleted, the affinity between the antibody and human FcγRIIIa is markedly reduced. It is known that the ADCC activity of an antibody containing a human Fc region is associated with the affinity (binding) between the Fc region and human FcγRIIIa on the surface of immune cells (Nordstrom. J, L, et al. , Breast Cancer Res., 13, 6, (2011)). Therefore, by evaluating the affinity between the antibody containing the human Fc region and human FcγRIIIa, the ADCC activity of the antibody to be produced can be monitored, and the process of culturing the mammalian cells capable of expressing the antibody can be monitored. As an example, when the affinity between the obtained antibody containing the human Fc region and human FcγRIIIa is decreased, the ADCC activity of the antibody is decreased, so that the ADCC activity of the antibody is improved, the manganese ion is increased. Medium components such as concentration and culture conditions (carbon dioxide concentration, temperature, pH, time, etc.) are adjusted appropriately.
ヒトFc領域を含む抗体とヒトFcγRIIIaとの親和性評価の好ましい態様として、ヒトFc領域を含む抗体とヒトFcγRIIIa固定化分離剤との結合力に基づく評価があげられる。ヒトFcγRIIIaを担体に固定化して得られるヒトFcγRIIIa固定化分離剤を充填したカラムに、ヒトFc領域を含む抗体をアプライすると、前記抗体が付加した糖鎖構造の違いに基づき分離され(特開2015−086216号公報、WO2018/150973号)、かつ前記抗体が有するADCC活性の違いに基づき分離される(特開2016−023152号公報、WO2018/150973号)。従って、前記分離パターンの形状に基づき、本発明における、抗体を発現可能な哺乳動物細胞の培養状態(培養工程)のモニタリングができる。具体的には、ヒトFcγRIIIa固定化分離剤を充填したカラムを用いてヒトFc領域を含む抗体を分離すると、ADCC活性が高い抗体がADCC活性が低い抗体よりも遅れて溶出される(すなわちヒトFcγRIIIa固定化分離剤との結合力が強い)。従って、前記分離により得られた溶出パターンのピーク面積またはピーク高さから、ADCC活性が高い抗体が溶出されるピーク(画分)の量および/または割合を算出し、当該量および/または割合が低下した場合、当該抗体が有するADCC活性が低下しているため、当該抗体が有するADCC活性を向上すべく、マンガンイオン濃度などの培地成分や培養条件(二酸化炭素濃度、溶存酸素濃度、温度、pH、時間など)を適宜調整する。 A preferred embodiment of the affinity evaluation between an antibody containing a human Fc region and human FcγRIIIa is an evaluation based on the binding force between an antibody containing a human Fc region and a human FcγRIIIa-immobilized separating agent. When an antibody containing a human Fc region is applied to a column filled with a human FcγRIIIa-immobilized separating agent obtained by immobilizing human FcγRIIIa on a carrier, the antibody is separated based on the difference in sugar chain structure added to the antibody (JP-A-2015). No. 086216, WO2018/150973) and based on the difference in ADCC activity of the antibody (Japanese Patent Laid-Open No. 2016-023152, WO2018/150973). Therefore, based on the shape of the separation pattern, the culture state (culture step) of the mammalian cells capable of expressing the antibody in the present invention can be monitored. Specifically, when a human Fc region-containing antibody is separated using a column packed with a human FcγRIIIa-immobilized separating agent, an antibody with high ADCC activity elutes later than an antibody with low ADCC activity (ie, human FcγRIIIa). Strong binding force with immobilized separating agent). Therefore, from the peak area or peak height of the elution pattern obtained by the separation, the amount and/or ratio of the peak (fraction) at which the antibody with high ADCC activity is eluted is calculated, and the amount and/or ratio is calculated. If it decreases, the ADCC activity of the antibody is decreased, so in order to improve the ADCC activity of the antibody, medium components such as manganese ion concentration and culture conditions (carbon dioxide concentration, dissolved oxygen concentration, temperature, pH) , Time, etc.) are adjusted appropriately.
また、培養条件や細胞株を同条件とし培地成分のみを変えて培養を行なうことで、よりADCC活性が高くなる培地成分の評価を行なうことができる。具体的には異なる培地で培養することで得られた抗体を前記ヒトFcγRIIIa固定化分離剤により評価を行ない、得られた分離パターンの形状やピーク(画分)の量および/または割合に基づき得られた抗体のADCC活性を比較することで培地の抗体に対する評価を行なう。 In addition, by culturing under the same culture conditions and cell lines and changing only the medium components, it is possible to evaluate the medium components having a higher ADCC activity. Specifically, the antibody obtained by culturing in a different medium is evaluated by the above-mentioned human FcγRIIIa-immobilized separating agent, and the antibody is obtained based on the shape of the obtained separation pattern and the amount and/or ratio of peaks (fractions). The ADCC activity of the obtained antibodies is compared to evaluate the medium for the antibody.
なお本明細書においてヒトFcγRIIIaとは、
(A)ヒトFcγRIIIa(UniProt No.P08637)のアミノ酸配列のうち17番目のグリシンから192番目のグルタミンまでのアミノ酸残基を少なくとも含む、Fc結合性タンパク質、または
(B)ヒトFcγRIIIa(UniProt No.P08637)のアミノ酸配列のうち17番目のグリシンから192番目のグルタミンまでのアミノ酸残基を少なくとも含み、ただし当該17番目から192番目までのアミノ酸残基において、1以上のアミノ酸残基が欠失、他のアミノ酸残基に置換、または付加されたポリペプチドを含む、Fc結合性タンパク質、
のことを意味する。また前記(B)の好ましい態様として、
特開2015−086216号公報で開示のFc結合性タンパク質、
特開2016−169197号公報で開示のFc結合性タンパク質、
特開2017−118871号公報で開示のFc結合性タンパク質、
WO2018/150973号で開示のFc結合性タンパク質、
WO2019/083048号で開示のFc結合性タンパク質、
があげられる。
In this specification, human FcγRIIIa means
(A) An Fc-binding protein containing at least an amino acid residue from glycine at position 17 to glutamine at position 192 in the amino acid sequence of human FcγRIIIa (UniProt No. P08637), or (B) human FcγRIIIa (UniProt No. P08637). ) At least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence, provided that one or more amino acid residues in the 17th to 192nd amino acid residues are deleted, An Fc-binding protein comprising a polypeptide substituted or added to an amino acid residue,
Means that. Further, as a preferable mode of the above (B),
Fc binding protein disclosed in JP-A-2005-086216,
Fc binding protein disclosed in JP-A-2016-169197,
Fc-binding protein disclosed in JP-A-2017-118871,
An Fc binding protein disclosed in WO2018/150973,
An Fc binding protein disclosed in WO2019/083048,
Can be given.
また本発明において、ADCC活性が向上した抗体とは、例えば、ヒトFcγRIIIa固定化分離剤を充填したカラムを用いた分離により得られた結果(溶出パターン)のうち、ADCC活性の高い抗体が位置するピーク面積またはピーク高さの割合が、マンガンイオン無添加培地で培養したときの前記割合と比較し、2%以上、好ましくは5%以上、より好ましくは10%以上、さらに好ましくは15%以上、さらにより好ましくは20%以上向上した抗体のことを意味する。 In the present invention, the antibody with improved ADCC activity refers to, for example, an antibody with high ADCC activity in the results (elution pattern) obtained by separation using a column packed with a human FcγRIIIa-immobilized separating agent. The ratio of the peak area or the peak height is 2% or more, preferably 5% or more, more preferably 10% or more, still more preferably 15% or more, as compared with the above ratio when cultured in a manganese ion-free medium. Even more preferably, it means an antibody with an improvement of 20% or more.
本発明は、抗体を発現可能な哺乳動物細胞を培養する工程と得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収する工程とを含む抗体の製造方法において、前記培養工程を0.01μMから50μMのマンガンイオンを添加した培地で行なうことを特徴としている。本発明により、抗癌剤など抗体依存性細胞障害活性を必要とする抗体を効率的に製造できる。 The present invention provides a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture, wherein the culturing step is performed. Is carried out in a medium supplemented with 0.01 μM to 50 μM manganese ion. According to the present invention, an antibody that requires antibody-dependent cellular cytotoxicity such as an anticancer agent can be efficiently produced.
また本発明で製造する抗体がヒトFc領域を含む抗体の場合、前記抗体とヒトFcγRIIIaとの親和性を評価することで、本発明の製造方法における培養工程のモニタリングや培地成分の評価が行なえる。 When the antibody produced in the present invention is an antibody containing a human Fc region, the affinity between the antibody and human FcγRIIIa can be evaluated to monitor the culture step and evaluate the medium components in the production method of the present invention. ..
以下、実施例および比較例を用いて、本発明をさらに詳細に説明するが、本発明はこれら例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples.
実施例1 抗インターロイキン6レセプター(IL−6R)抗体発現細胞の構築
(1)以下の方法で抗IL−6R抗体を哺乳動物細胞で発現可能なベクターを構築した。
(1−1)配列番号1に記載のジヒドロ葉酸レダクターゼ(dihydrofolate reductase、dhfr)およびSV40のPolyAをコードする遺伝子に制限酵素SacII認識配列列(CCGCGG)を5’末端および3’末端の両方に付加した遺伝子を全合成し(Integrated DNA Technologies社に委託)プラスミドにクローニングした。
(1−2)(1−1)で作製したプラスミドで大腸菌JM109株を形質転換した。得られた形質転換体を培養し、プラスミドを抽出したのち、制限酵素SacIIで消化することで、dhfr−SV40PolyAをコードする遺伝子を調製しdhfr−P1と命名した。
(1−3)pIRESベクター(Clontech社製)を鋳型として、配列番号2(5’−TCC[CCGCGG]GCGGGACTCTGGGGTTCGAAATGACCG−3’)および配列番号3(5’−TCC[CCGCGG]GGTGGCTCTAGCCTTAAGTTCGAGACTG−3’)に記載の配列からなるオリゴヌクレオチドプライマー(配列番号2および3中の角かっこは制限酵素SacII認識配列を示している)を用いてPCRを行なった。具体的には、表1に示す組成の反応液を調製し、当該反応液を98℃で30秒間熱処理後、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で5分間の第3ステップを1サイクルとする反応を25サイクル繰り返ことで実施した。このPCRにより、pIRESベクターのうちネオマイシン耐性遺伝子を除いた領域を増幅した。
Example 1 Construction of cells expressing anti-interleukin 6 receptor (IL-6R) antibody (1) A vector capable of expressing anti-IL-6R antibody in mammalian cells was constructed by the following method.
(1-1) Addition of a restriction enzyme SacII recognition sequence sequence (CCGCGG) to both the 5'end and the 3'end of the gene encoding dihydrofolate reductase (dihydrofolate reductase, dhfr) shown in SEQ ID NO: 1 and PolyA of SV40. The gene was totally synthesized (consigned to Integrated DNA Technologies) and cloned into a plasmid.
(1-2) Escherichia coli JM109 strain was transformed with the plasmid prepared in (1-1). The resulting transformant was cultured, the plasmid was extracted, and then the gene encoding dhfr-SV40PolyA was prepared by digesting with the restriction enzyme SacII, and named as dhfr-P1.
(1-3) Using the pIRES vector (manufactured by Clontech) as a template, SEQ ID NO: 2 (5′-TCC[CCGCGG]GCGGGACTCTGGGGTCTGAAATGACCG-3′) and SEQ ID NO:3 (5′-TCC[CCGCGG]GGTGGGCTCTAGCCTTAAGTTCGAGACTG-3′). PCR was carried out using an oligonucleotide primer having the described sequence (the brackets in SEQ ID NOS: 2 and 3 represent the restriction enzyme SacII recognition sequence). Specifically, a reaction solution having the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 98° C. for 30 seconds, followed by the first step at 98° C. for 10 seconds, the second step at 55° C. for 5 seconds, and the second step at 72° C. The reaction was carried out by repeating 25 cycles of the third step of 1 minute at 5° C. for 25 cycles. By this PCR, the region excluding the neomycin resistance gene in the pIRES vector was amplified.
(1−4)(1−3)で作製したPCR産物を精製後、制限酵素SacIIで消化し、(1−2)で調製したdhfr−P1とライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することでdhfr遺伝子を含んだ発現ベクターpIRES−dhfrを得た。 (1-4) After purifying the PCR product prepared in (1-3), it was digested with the restriction enzyme SacII and ligated with dhfr-P1 prepared in (1-2). Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain an expression vector pIRES-dhfr containing the dhfr gene.
(2)(1)で作製したpIRES−dhfrを鋳型として配列番号4(5’−TTTAAATCA[GCGGCCGC]GCAGCACCATGGCCTGAAATAACCTCTG−3’)および配列番号5(5’−GCAAGTAAAACCTCTACAAATGTGGTAAA[CGATCG]CTCCGGTGCCCGTー3’)に記載の配列からなるオリゴヌクレオチドプライマー(配列番号4中の角かっこは制限酵素NotI認識配列を、配列番号5中の角かっこは制限酵素PvuI認識配列を、それぞれ示している)を用いてPCRを行なった。具体的には、表2に示す組成の反応液を調製し、当該反応液を98℃で1分間熱処理後、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で1分間の第3ステップを1サイクルとする反応を30サイクル繰り返すことで実施した。このPCRにより増幅したPCR産物(SV40プロモーター、dhfr、SV40のPolyAまでの領域を)をdhfr−P2と命名した。 (2) Using the pIRES-dhfr prepared in (1) as a template, SEQ ID NO: 4 (5′-TTTAAATCA[GCGGCCGC]GCAGCACCATGGCCTGAAATAACCTCTTG-3′) and SEQ ID NO:5 (5′-GCAAGTAAAACCTCTACAAATCGTGTAAA]CGTGCTAGA[CGTGCTAGAG]CGTGCTAGAG] PCR was performed using an oligonucleotide primer having the sequence of SEQ ID NO: 4 (the brackets in SEQ ID NO: 4 represent the restriction enzyme NotI recognition sequence, and the brackets in SEQ ID NO: 5 represent the restriction enzyme PvuI recognition sequence). .. Specifically, a reaction solution having the composition shown in Table 2 was prepared, the reaction solution was heat-treated at 98° C. for 1 minute, and then the first step was performed at 98° C. for 10 seconds, the second step was performed at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1° C. as one cycle. The PCR product (SV40 promoter, dhfr, the region up to PolyA of SV40) amplified by this PCR was designated as dhfr-P2.
(3)ヒト抗体の重鎖定常領域を含んだpFUSEss−CHIg−hG1(InvivoGen社製)、ヒト抗体の軽鎖定常領域を含んだpFUSE2ss−CLIg−hk(InvivoGen社製)および(2)で作製したdhfr−P2をそれぞれ制限酵素NotIおよびPvuIで消化した後、精製しライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することでSV40プロモーター、dhfr、SV40のPolyAを含んだpFUSEss−CHIg−hG1およびpFUSE2ss−CLIg−hkを得た。pFUSEss−CHIg−hG1にSV40プロモーター、dhfrおよびSV40のPolyAを組込んだプラスミドをpFU−CHIg−dhfrと命名し、pFUSE2ss−CLIg−hkにSV40プロモーター、dhfrおよびSV40のPolyAを組込んだプラスミドをpFU−CLIg−dhfrと命名した。 (3) pFUSEss-CHIg-hG1 (manufactured by InvivoGen) containing a heavy chain constant region of a human antibody, pFUSE2ss-CLIg-hk (manufactured by InvivoGen) containing a human antibody light chain constant region, and (2) The digested dhfr-P2 was digested with restriction enzymes NotI and PvuI, respectively, and then purified and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hk containing SV40 promoter, dhfr, and PolyA of SV40. A plasmid in which SV40 promoter, dhfr and PolyA of SV40 were incorporated into pFUSEss-CHIg-hG1 was named pFU-CHIg-dhfr, and a plasmid in which SV40 promoter, dhfr and PolyA of SV40 were incorporated into pFUSE2ss-CLIg-hk was pFU. It was named -CLIg-dhfr.
(4)配列番号6に記載のアミノ酸配列からなる抗インターロイキン6レセプター(以下、IL−6R)抗体の重鎖可変領域をコードする配列番号7に記載のポリヌクレオチドの5’末端に制限酵素EcoRI認識配列(GAATTC)とフレームシフト抑制のためグアニン(G)を付加し、3’末端に制限酵素NheI認識配列(GCTAGC)を付加した遺伝子を全合成しプラスミドにクローニングした(FASMAC社に委託)。作製したプラスミドをpUC−VH6Rと命名した。また、配列番号8に記載のアミノ酸配列からなる抗IL−6R抗体の軽鎖可変領域をコードする配列番号9に記載のポリヌクレオチドの5‘末端に制限酵素EcoRI認識配列(GAATTC)とフレームシフト抑制のためグアニン(G)を付加し、3’末端に制限酵素BsiWI認識配列(CGTACG)を付加した遺伝子を全合成しプラスミドにクローニングした(FASMAC社に委託)。作製したプラスミドをpUC−VL6Rと命名した。 (4) Restriction enzyme EcoRI at the 5'end of the polynucleotide of SEQ ID NO: 7 encoding the heavy chain variable region of the anti-interleukin 6 receptor (hereinafter IL-6R) antibody consisting of the amino acid sequence of SEQ ID NO: 6 A gene having a recognition sequence (GAATTC) and guanine (G) added to suppress frame shift and a restriction enzyme NheI recognition sequence (GCTAGC) added to the 3'end was totally synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was named pUC-VH6R. In addition, a restriction enzyme EcoRI recognition sequence (GAATTC) and a frame shift inhibitor are added to the 5'end of the polynucleotide shown in SEQ ID NO: 9 encoding the light chain variable region of the anti-IL-6R antibody consisting of the amino acid sequence shown in SEQ ID NO: 8. Therefore, a gene in which guanine (G) was added and a restriction enzyme BsiWI recognition sequence (CGTACG) was added to the 3'end was totally synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was named pUC-VL6R.
(5)(4)で作製したpUC−VH6Rおよび(3)で作製したpFU−CHIg−dhfrをそれぞれ制限酵素EcoRI、NheIで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗IL−6R抗体の重鎖(H鎖)を発現するプラスミドpFU−6RH−dhfrを得た。また(4)で作製したpUC−VL6Rおよび(3)で作製したpFU−CLIg−dhfrをそれぞれ制限酵素EcoRI、BsiWIで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗IL−6R抗体の軽鎖(L鎖)を発現するプラスミドpFU−6RL−dhfrを得た。 (5) The pUC-VH6R prepared in (4) and the pFU-CHIg-dhfr prepared in (3) were digested with restriction enzymes EcoRI and NheI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-6RH-dhfr expressing the heavy chain (H chain) of the anti-IL6R antibody. Further, pUC-VL6R prepared in (4) and pFU-CLIg-dhfr prepared in (3) were digested with restriction enzymes EcoRI and BsiWI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-6RL-dhfr expressing the light chain (L chain) of the anti-IL-6R antibody.
実施例2 抗IL−6R抗体高発現細胞の構築
(1)実施例1で作製したpFU−6RH−dhfrおよびpFU−6RL−dhfrを、CHO細胞(DG44株)にNeon Transfection System(Thermo Fisher Scientific社製)を用いて遺伝子導入した。その後、50μg/mLのカナマイシン、40mL/LのGlutaMAX(Thermo Fisher Scientific社製)を含んだCD OptiCHO Medium(Thermo Fisher Scientific社製)で形質転換細胞を培養抗IL−6R抗体発現細胞を得た。その後、培地に50ng/mLのメトトレキサート
(MTX)を添加することで遺伝子増幅を行なった。
Example 2 Construction of cells with high expression of anti-IL-6R antibody (1) pFU-6RH-dhfr and pFU-6RL-dhfr prepared in Example 1 were used as CHO cells (DG44 strain) in the Neoon Transfection System (Thermo Fisher Scientific). Gene transfer was carried out using Then, transformed cells were cultured with CD OptiCHO Medium (manufactured by Thermo Fisher Scientific) containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX (manufactured by Thermo Fisher Scientific) to obtain anti-IL-6R antibody-expressing cells. Then, gene amplification was performed by adding 50 ng/mL methotrexate (MTX) to the medium.
(2)(1)でMTX処理をした細胞を限外希釈法により単クローン化し、下記に記載のELISA(Enzyme−Linked ImmunoSorbent Assay)を用いて、抗IL−6R抗体を安定的に高生産可能な細胞を選択した。
(2−1)抗ヒトFab抗体(Bethyl社製)を、96穴マイクロプレートのウェルに1μg/wellで固定化した(4℃で一晩)。固定化終了後、2%(w/v)のSKIM MILK(Becton Dickinson社製)および150mM塩化ナトリウムを含んだ20mMのトリス塩酸緩衝液(pH7.4)によりブロッキングした。
(2−2)洗浄緩衝液(0.05%[w/v]のTween 20(商品名)と150mMのNaClとを含む20mM Tris−HCl緩衝液(pH8.0))で洗浄後、抗体を含んだ培養上清を添加し、抗体と固定化タンパク質とを反応させた(30℃で1時間)。
(2−3)反応終了後、前記洗浄緩衝液で洗浄し、100ng/mLに希釈したペルオキシターゼで標識された抗ヒトFc抗体(Bethyl社製)を100μL/wellで添加した。
(2−4)30℃で1時間反応し、前記洗浄緩衝液で洗浄した後、TMB Peroxidase Substrate(KPL社製)を50μL/wellで添加した。その後、1Mのリン酸を50μL/wellで添加することで発色を止め、マイクロプレートリーダー(テカン社製)を用いて450nmの吸光度を測定し、測定値の高い抗IL−6R抗体高生産細胞株を選択した。
(2) The cells treated with MTX in (1) can be monocloned by the limiting dilution method, and stable high production of anti-IL-6R antibody can be achieved by using the following ELISA (Enzyme-Linked Immunosorbent Assay). Cells were selected.
(2-1) An anti-human Fab antibody (manufactured by Bethyl) was immobilized in wells of a 96-well microplate at 1 μg/well (4° C. overnight). After completion of immobilization, blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 2% (w/v) SKIM MILK (manufactured by Becton Dickinson) and 150 mM sodium chloride.
(2-2) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 8.0) containing 0.05% [w/v] Tween 20 (trade name) and 150 mM NaCl), the antibody was washed. The containing culture supernatant was added to react the antibody with the immobilized protein (30° C. for 1 hour).
(2-3) After the reaction was completed, the reaction mixture was washed with the washing buffer, and an anti-human Fc antibody labeled with peroxidase (manufactured by Bethyl) diluted to 100 ng/mL was added at 100 μL/well.
(2-4) After reacting at 30° C. for 1 hour and washing with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added at 50 μL/well. After that, color development was stopped by adding 1 M phosphoric acid at 50 μL/well, the absorbance at 450 nm was measured using a microplate reader (manufactured by Tecan), and a cell line with high production of anti-IL-6R antibody was measured. Was selected.
(3)MTX濃度を段階的(50nM、500nM、1μM、2μM、4μM、8μM、16μM、32μM、64μM)に上昇させながら、限外希釈を行ない(2)に記載のELISAでクローン選択を行なうことを繰り返した。その結果、抗IL−6R抗体高生産細胞株を得た。 (3) Perform clonal selection by the ELISA described in (2) while increasing the MTX concentration stepwise (50 nM, 500 nM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM). Was repeated. As a result, a cell line with high production of anti-IL-6R antibody was obtained.
実施例3 抗ヒトgp130受容体(gp130R)抗体高発現細胞の構築
(1)配列番号10に記載のアミノ酸配列からなる抗ヒトgp130受容体(gp130R)抗体の重鎖可変領域をコードする配列番号11に記載のポリヌクレオチドの5’末端に制限酵素EcoRI認識配列(GAATTC)とフレームシフト抑制のためグアニン(G)を付加し、3’末端に制限酵素NheI認識配列(GCTAGC)を付加した遺伝子を全合成しプラスミドにクローニングした(FASMAC社に委託)。作製したプラスミドをpUC−VHgp130と命名した。また、配列番号12に記載のアミノ酸配列からなる抗ヒトgp130R抗体の軽鎖可変領域をコードする配列番号13に記載のポリヌクレオチドの5’末端に制限酵素EcoRI認識配列(GAATTC)とフレームシフト抑制のためグアニン(G)を付加し、3’末端に制限酵素BsiWI認識配列(CGTACG)を付加した遺伝子を全合成しプラスミドにクローニングした(FASMAC社に委託)。作製したプラスミドをpUC−VLgp130と命名した。
Example 3 Construction of anti-human gp130 receptor (gp130R) antibody highly expressing cells (1) SEQ ID NO: 11 encoding heavy chain variable region of anti-human gp130 receptor (gp130R) antibody having the amino acid sequence set forth in SEQ ID NO: 10 The polynucleotide having the restriction enzyme EcoRI recognition sequence (GAATTC) and guanine (G) added for frameshift suppression at the 5'end and the 3'terminal addition of the restriction enzyme NheI recognition sequence (GCTAGC) at the 5'end Synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was designated as pUC-VHgp130. In addition, a restriction enzyme EcoRI recognition sequence (GAATTC) at the 5'end of the polynucleotide shown in SEQ ID NO: 13 encoding the light chain variable region of the anti-human gp130R antibody consisting of the amino acid sequence shown in SEQ ID NO: 12 and a frameshift inhibitor Therefore, a gene in which guanine (G) was added and a restriction enzyme BsiWI recognition sequence (CGTACG) was added to the 3'end was totally synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was named pUC-VLgp130.
(2)(1)で作製したpUC−VHgp130および実施例1(3)で作製したpFU−CHIg−dhfrをそれぞれ制限酵素EcoRI、NheIで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗ヒトgp130R抗体の重鎖(H鎖)を発現するプラスミドpFU−gp130H−dhfrを得た。また(1)で作製したpUC−VLgp130および実施例1(3)で作製したpFU−CLIg−dhfrをそれぞれ制限酵素EcoRI、BsiWIで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗ヒトgp130R抗体の軽鎖(L鎖)を発現するプラスミドpFU−gp130L−dhfrを得た。 (2) pUC-VHgp130 produced in (1) and pFU-CHIg-dhfr produced in Example 1(3) were digested with restriction enzymes EcoRI and NheI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-gp130H-dhfr expressing the heavy chain (H chain) of the anti-human gp130R antibody. In addition, pUC-VLgp130 prepared in (1) and pFU-CLIg-dhfr prepared in Example 1 (3) were digested with restriction enzymes EcoRI and BsiWI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-gp130L-dhfr expressing the light chain (L chain) of the anti-human gp130R antibody.
(3)(2)で得られたpFU−gp130L−dhfrを鋳型として、配列番号14(5’−GCCTCTTCCCGGGCCGAGCTGGTGCTGACTC−3’)および配列番号15(5’−AAT[GCGGCCGC]TACTAACACTCTCCCCTGTTGAAGC−3’)(配列番号15中の角かっこは制限酵素NotI認識配列を示している)に記載の配列からなるオリゴヌクレオチドプライマーを用いてPCRを行なった。具体的には、表3に示す組成の反応液を調製し、当該反応液を98℃で5分間熱処理後、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で15分間の第3ステップを1サイクルとする反応を30サイクル繰り返すことで実施した。このPCRにより抗ヒトgp130R抗体のL鎖全長をコードする遺伝子を増幅し、精製後に得られたPCR産物をhgp130−L1と命名した。 (3) Using the pFU-gp130L-dhfr obtained in (2) as a template, SEQ ID NO: 14 (5'-GCCTCTTCCCGGGCCGAGCTGGGTGCTGAACTC-3') and SEQ ID NO: 15 (5'-AAT[GCGGCCGC]TACTAACACTCTCCCCCTGTGAAGC-3'). PCR was carried out using an oligonucleotide primer having the sequence described in "(15) shows the restriction enzyme NotI recognition sequence". Specifically, a reaction solution having the composition shown in Table 3 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 15° C. for 30 cycles. A gene encoding the full-length L chain of the anti-human gp130R antibody was amplified by this PCR, and the PCR product obtained after purification was named hgp130-L1.
(4)(3)で得られたhgp130−L1を鋳型として、配列番号16(5’−CTA[GAATTC]GCCACCATGACCCGGCTGACC−3’)(配列番号16中の角かっこは制限酵素EcoRI認識配列を示している)および配列番号15に記載の配列からなるオリゴヌクレオチドプライマーを用いて、配列番号17に記載のアミノ酸配列からなるシグナル配列(MTRLTVLALLAGLLASSRA)を付加するため、配列番号17に記載のアミノ酸配列をコードする配列番号18(5’−ATGACCCGGCTGACCGTGCTGGCCCTGCTGGCTGGCCTGCTCGCCTCTTCCCGGGCC−3’)に記載のポリヌクレオチドを添加してPCRを行なった。具体的には、表4に示す組成の反応液を調製し、当該反応液を98℃で5分間熱処理後、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で1.5分間の第3ステップを1サイクルとする反応を30サイクル繰り返すことで実施した。このPCRにより、抗ヒトgp130R抗体のL鎖全長をコードする遺伝子に配列番号17に記載のシグナル配列をコードする遺伝子を付加した遺伝子を増幅し、精製後に得られたPCR産物をhgp130−L2と命名した。 (4) Using the hgp130-L1 obtained in (3) as a template, SEQ ID NO: 16 (5'-CTA [GAATTC] GCCACCCATGACCCGGCTGACC-3') (the brackets in SEQ ID NO: 16 indicate the restriction enzyme EcoRI recognition sequence). And a signal sequence (MTRLTVLLALLGLASSRA) consisting of the amino acid sequence of SEQ ID NO: 17 is added using an oligonucleotide primer consisting of the sequence of SEQ ID NO: 15 to encode the amino acid sequence of SEQ ID NO: 17. PCR was carried out by adding the polynucleotide described in SEQ ID NO: 18 (5′-ATGACCCCGGCTGACCGTGCTGGCCCCTGCTGCTGGCCTGTCCGCCCTCTCCCGGGCC-3′). Specifically, a reaction solution having the composition shown in Table 4 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1.5° C. for 30 cycles. A gene obtained by adding the gene encoding the signal sequence of SEQ ID NO: 17 to the gene encoding the full-length L chain of the anti-human gp130R antibody was amplified by this PCR, and the PCR product obtained after purification was named hgp130-L2. did.
(5)(4)で得られたhgp130−L2および図1に記載の発現ベクターpEFdをそれぞれ制限酵素EcoRIおよびNotIで消化し、精製後ライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することで哺乳動物細胞において抗ヒトgp130R抗体のL鎖を発現可能なpEFd−gp130Lを得た。なお、図1に記載のpEFdベクターは配列番号19に記載のEF1αプロモーターのうち621番目のグアニンから798番目のグアニンまでのヌクレオチドが欠損しているプロモーター(EFd pro)を有しており、SV40のpolyAおよびdhfr遺伝子も有している(特開2019−106976号公報)。 (5) The hgp130-L2 obtained in (4) and the expression vector pEFd shown in FIG. 1 were digested with restriction enzymes EcoRI and NotI, respectively, and purified and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain pEFd-gp130L capable of expressing the L chain of the anti-human gp130R antibody in mammalian cells. The pEFd vector shown in FIG. 1 has a promoter (EFd pro) in which the nucleotides from the 621st guanine to the 798th guanine of the EF1α promoter shown in SEQ ID NO: 19 are deleted, and the pEFd vector of SV40 It also has polyA and dhfr genes (JP-A-2019-106976).
(6)(2)で得られたpFU−gp130H−dhfrを鋳型として、配列番号20(5’−GCCTCTTCCCGGGCCCAGGTTCAACTCCAG−3’)および配列番号21(5’−AAT[GCGGCCGC]TATCATTTACCCGGAGACAGGGAGAG−3’)(配列番号21中の角かっこは制限酵素NotI認識配列を示している)に記載の配列からなるオリゴヌクレオチドプライマーを用いてPCRを行なった。具体的には、表3に示す組成の反応液を調製し、当該反応液を98℃で5分間熱処理後、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で1.5分間の第3ステップを1サイクルとする反応を30サイクル繰り返すことで実施した。このPCRにより、抗ヒトgp130R抗体のH鎖全長をコードする遺伝子を増幅し精製後に得られたPCR産物をhgp130−H1と命名した。 (6) SEQ ID NO: 20 (5′-GCCCTTCCCCGGGCCCAGGTTCAACTCCAG-3′) and SEQ ID NO: 21 (5′-AAT[GCGGCCGC]TATCATTTTACCCGGAGACAGGGAGAG-3′) using the pFU-gp130H-dhfr obtained in (2) as a template. PCR was carried out using an oligonucleotide primer having the sequence described in (21) indicates the restriction enzyme NotI recognition sequence. Specifically, a reaction solution having the composition shown in Table 3 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1.5° C. for 30 cycles. The PCR product obtained by amplifying the gene encoding the full-length H chain of the anti-human gp130R antibody by this PCR and purifying was named hgp130-H1.
(7)(6)で得られたhgp130−H1を鋳型として、配列番号16および配列番号21に記載の配列からなるオリゴヌクレオチドプライマーを用いて、配列番号17に記載のアミノ酸配列からなるシグナル配列を付加するため、配列番号18に記載のポリヌクレオチドを添加してPCRを行なった。具体的には、表4に示す組成の反応液を調製し、当該反応液を98℃で5分間熱処理後、98℃で10秒間の第1ステップ、55℃で5秒間の第2ステップ、72℃で1.5分間の第3ステップを1サイクルとする反応を30サイクル繰り返すことで実施した。このPCRにより、抗ヒトgp130R抗体のH鎖全長をコードする遺伝子に配列番号17に記載のシグナル配列をコードする遺伝子を付加した遺伝子を増幅し、精製後に得られたPCR産物をhgp130−H2と命名した。 (7) Using the hgp130-H1 obtained in (6) as a template and an oligonucleotide primer consisting of the sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 21, a signal sequence consisting of the amino acid sequence set forth in SEQ ID NO: 17 was obtained. To add, the polynucleotide of SEQ ID NO: 18 was added and PCR was performed. Specifically, a reaction solution having the composition shown in Table 4 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1.5° C. for 30 cycles. A gene obtained by adding the gene encoding the signal sequence of SEQ ID NO: 17 to the gene encoding the full-length H chain of the anti-human gp130R antibody was amplified by this PCR, and the PCR product obtained after purification was named hgp130-H2. did.
(8)(7)で得られたhgp130−H2および図1に記載の発現ベクターpEFdをそれぞれ制限酵素EcoRIおよびNotIで消化し、精製後ライゲーションした。当該ライゲーション産物で大腸菌JM109株を形質転換し、培養した形質転換体からプラスミドを抽出することで哺乳動物細胞において抗ヒトgp130R抗体を発現可能なpEFd−gp130Hを得た。 (8) The hgp130-H2 obtained in (7) and the expression vector pEFd shown in FIG. 1 were digested with restriction enzymes EcoRI and NotI, respectively, and purified and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain pEFd-gp130H capable of expressing anti-human gp130R antibody in mammalian cells.
(9)(5)で得られたpEFd−gp130L、(8)で得られたpEFd−gp130Hを用いた以外は実施例2と同様の方法で高発現細胞の構築を行ない、MTX濃度を50nM、250nM、1μMと段階的にあげることで抗ヒトgp130R抗体高発現細胞を構築した。 (9) High expression cells were constructed in the same manner as in Example 2 except that pEFd-gp130L obtained in (5) and pEFd-gp130H obtained in (8) were used, and MTX concentration was 50 nM. Anti-human gp130R antibody high expressing cells were constructed by stepwise increasing to 250 nM and 1 μM.
実施例4 フラスコ培養におけるマンガンイオン添加の効果(その1)
(1)実施例2で得られた抗IL−6R抗体高発現細胞または実施例3で得られた抗ヒトgp130R抗体高発現細胞を、50μg/mLのカナマイシン、40mL/LのGlutaMAX(Thermo Fisher Scientific社製)を含む20mLのBalanCD CHO Growth A medium(Irvine Scientific社製)を入れた125mLの三角フラスコ(Corning社製)に接種し、130rpm、37℃、8%CO2の条件下で振盪培養した。
Example 4 Effect of Manganese Ion Addition on Flask Culture (Part 1)
(1) The anti-IL-6R antibody high-expressing cells obtained in Example 2 or the anti-human gp130R antibody high-expressing cells obtained in Example 3 were mixed with 50 μg/mL kanamycin and 40 mL/L GlutaMAX (Thermo Fisher Scientific). (Manufactured by the company) containing 20 mL of BalanCD CHO Growth A medium (manufactured by Irvine Scientific) was inoculated into a 125 mL Erlenmeyer flask (manufactured by Corning), and shake-cultured under conditions of 130 rpm, 37° C. and 8% CO 2 . ..
(2)細胞数が1×106cells/mL以上になったところで、50μg/mLのカナマイシン、40mL/LのGlutaMAXを含んだBalanCD CHO Growth A mediumに0.2×106cells/mLとなるように前記細胞を接種し、フィルター滅菌した50mMの硫酸マンガンを終濃度で0μM(添加せず)、0.01μM、0.1μM、1μM、5μM、10μMもしくは50μMとなるように添加し、総量を20mLに調製して125mLの三角フラスコで、130rpm、37℃、8%CO2の条件下で10日間、振盪培養した。培養途中で培養液をサンプリングし、生細胞数をCountess(Thermo Fisher Scientific社製)を使用して測定し、抗体生産性はヒトIgG1(シグマ社製)を標準品とした検量線に基づき実施例2(2)に記載のELISA法にて測定した。 (2) When the cell number becomes 1×10 6 cells/mL or more, 0.2×10 6 cells/mL is obtained in BalanCD CHO Growth A medium containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX. The above cells were inoculated as described above, and 50 mM manganese sulfate filter-sterilized was added to a final concentration of 0 μM (no addition), 0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM or 50 μM, and the total amount was added. It was adjusted to 20 mL and shake-cultured in a 125 mL Erlenmeyer flask for 10 days under the conditions of 130 rpm, 37° C. and 8% CO 2 . The culture solution was sampled during the culture, and the number of viable cells was measured using Countess (Thermo Fisher Scientific), and the antibody productivity was determined based on a calibration curve using human IgG1 (Sigma) as a standard example. It was measured by the ELISA method described in 2(2).
(3)培養終了後の培養液を遠心分離によって細胞および不純物を除去し、得られた上清を、1.0mLのMabSelect SuRe LX(GEヘルスケア社製)をオープンカラムに詰めて作製した分離カラム(150mMの塩化ナトリウムを含んだ20mMのTris−HCl(pH7.4)で平衡化済)にアプライした。 (3) Cells and impurities were removed from the culture solution after completion of the culture by centrifugation, and the obtained supernatant was prepared by packing 1.0 mL of MabSelect SuRe LX (manufactured by GE Healthcare) into an open column. It was applied to a column (equilibrated with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride).
(4)前記平衡化に用いた緩衝液10mLで前記分離カラムを洗浄後、0.1Mのグリシン塩酸緩衝液(pH3.0)4mLで前記分離カラムに吸着した抗体を溶出した。溶出液に1mLの1M Tris−HCl(pH8.0)を加えることでpHを中性領域に戻し、限外ろ過膜で濃縮しながら50mMのクエン酸緩衝液(pH6.5)に緩衝液交換することで、培地に添加したマンガンイオン濃度が異なる、高純度な抗IL−6R抗体および抗ヒトgp130R抗体を得た。 (4) After washing the separation column with 10 mL of the buffer used for the equilibration, the antibody adsorbed on the separation column was eluted with 4 mL of 0.1 M glycine-hydrochloric acid buffer (pH 3.0). The pH is returned to the neutral range by adding 1 mL of 1 M Tris-HCl (pH 8.0) to the eluate, and the buffer is exchanged with a 50 mM citrate buffer (pH 6.5) while concentrating with an ultrafiltration membrane. Thus, high-purity anti-IL-6R antibody and anti-human gp130R antibody having different manganese ion concentrations added to the medium were obtained.
(5)ヒトFcγRIIIaを固定化した担体(分離剤)を詰めたカラム(FcR9_Fカラム、WO2018/150973号の実施例5に記載)を用いて、下記の方法により(4)で得られた抗IL−6R抗体および抗ヒトgp130R抗体を分析した。
(5−1)FcR9_Fカラムを高速液体クロマトグラフィー装置(島津製作所社製)に接続し、カラムオーブンで前記カラムを25℃の恒温状態に維持し、50mMのクエン酸緩衝液(pH6.5)を流速1.0mL/minで10分間流すことにより前記カラムを平衡化した。
(5−2)(4)で得た抗IL−6R抗体および抗ヒトgp130R抗体を(5−1)で用いた緩衝液で1.0mg/mLに希釈し、当該希釈抗体溶液を流速1.0mL/min
にて10μL添加した。
(5−3)(5−1)で用いた緩衝液を流速1.0mL/minで2分間流した後、50
mMのクエン酸緩衝液(pH4.5)によるpHグラジエント(18分間で50mMのク
エン酸緩衝液(pH4.5)が100%となるグラジエント)でFcR9_Fカラムに吸
着した抗体を溶出した。
(5) Using a column (FcR9_F column, described in Example 5 of WO2018/150973) packed with a carrier (separating agent) on which human FcγRIIIa is immobilized, the anti-IL obtained in (4) by the following method The -6R antibody and anti-human gp130R antibody were analyzed.
(5-1) The FcR9_F column was connected to a high performance liquid chromatography apparatus (manufactured by Shimadzu Corporation), the column was kept at a constant temperature of 25° C. in a column oven, and a 50 mM citrate buffer solution (pH 6.5) was used. The column was equilibrated by flowing at a flow rate of 1.0 mL/min for 10 minutes.
(5-2) The anti-IL-6R antibody and the anti-human gp130R antibody obtained in (4) were diluted to 1.0 mg/mL with the buffer solution used in (5-1), and the diluted antibody solution was flowed at a flow rate of 1. 0 mL/min
Then, 10 μL was added.
(5-3) After flowing the buffer solution used in (5-1) at a flow rate of 1.0 mL/min for 2 minutes, 50
The antibody adsorbed on the FcR9_F column was eluted with a pH gradient of mM citrate buffer (pH 4.5) (gradient at which 50 mM citrate buffer (pH 4.5) was 100% in 18 minutes).
培地中に添加したマンガンイオン濃度の違いによる、抗IL−6R抗体の生産性を比較した結果を図2に、抗IL−6R抗体高発現細胞数(生細胞数)の推移を図3に、抗IL−6R抗体のFcR9_Fカラムによる分析結果を図4に、抗gp130R抗体の生産性を比較した結果を図5に、抗gp130R抗体高発現細胞数(生細胞数)の推移を図6に、抗IL−6R抗体のFcR9_Fカラムによる分析結果を図7に、それぞれ示す。また図4に記載の分析結果にある各ピークの面積割合を表5に、図7に記載の分析結果にある各ピークの面積割合を表6に、それぞれ示す。 The results of comparing the productivity of the anti-IL6R antibody due to the difference in the manganese ion concentration added to the medium are shown in FIG. 2, and the transition of the number of highly expressing anti-IL-6R antibody cells (the number of viable cells) is shown in FIG. The results of analysis of the anti-IL-6R antibody by the FcR9_F column are shown in FIG. 4, the results of comparison of the productivity of the anti-gp130R antibody are shown in FIG. 5, the transition of the number of high-expressing anti-gp130R antibody cells (viable cell number) is shown in FIG. The analysis results of the anti-IL-6R antibody by the FcR9_F column are shown in FIG. 7, respectively. The area ratio of each peak in the analysis result shown in FIG. 4 is shown in Table 5, and the area ratio of each peak in the analysis result shown in FIG. 7 is shown in Table 6.
表5および表6より、抗IL−6R抗体高発現細胞、抗gp130R抗体高発現細胞数とも、培地に0.01μMから50μMのマンガンイオンを添加して培養すると、マンガンイオン未添加時と比較し、FcR9_Fカラムとの結合力が強いピーク(ピーク3)の割合が増加していることがわかる。ADCC活性の高い抗体ほど、FcγRIIIa固定化分離剤との結合力が強まる(特開2016−023152号公報、WO2018/150973号)。すなわち図4および図7におけるピーク3の示す割合が増加する。このことから、抗体を発現可能な哺乳動物細胞を培養する際、0.01μMから50μM(特に0.1μMから50μM)のマンガンイオンを添加した培地で培養することで、前記哺乳細胞が発現する抗体が有するADCC活性が向上することがわかる。 From Table 5 and Table 6, both anti-IL-6R antibody high-expressing cells and anti-gp130R antibody high-expressing cell numbers were compared with those when no manganese ion was added when the culture was performed by adding 0.01 μM to 50 μM manganese ion to the medium. It can be seen that the ratio of peaks (peak 3) having strong binding force with the FcR9_F column is increasing. The higher the ADCC activity of the antibody, the stronger the binding strength to the FcγRIIIa-immobilized separating agent (JP-A-2016-023152, WO2018/150973). That is, the ratio of peak 3 in FIGS. 4 and 7 increases. Therefore, when culturing mammalian cells capable of expressing antibodies, the antibodies expressed by the mammalian cells can be obtained by culturing in a medium containing manganese ions of 0.01 μM to 50 μM (particularly 0.1 μM to 50 μM). It can be seen that the ADCC activity possessed by is improved.
実施例5 フラスコ培養におけるマンガンイオン添加の効果(その2)
使用した培地をCD Opti CHO Medium(Thermo Fisher Scientific社製)とし、培養期間を11日間とした他は、実施例4と同様の方法を用いて、実施例2で得られた抗IL−6R抗体高発現細胞、および実施例3で得られた抗ヒトgp130R抗体高発現細胞を培養し、抗体生産性および生細胞数の測定、ならびに得られた培養物中に含まれる前記細胞が発現した抗体のFcR9_Fカラムによる分析を行なった。
Example 5 Effect of Manganese Ion Addition on Flask Culture (Part 2)
The anti-IL-6R antibody obtained in Example 2 was used in the same manner as in Example 4 except that the medium used was CD Opti CHO Medium (manufactured by Thermo Fisher Scientific) and the culture period was 11 days. The high-expressing cells and the anti-human gp130R antibody-high-expressing cells obtained in Example 3 were cultivated, the antibody productivity and the number of viable cells were measured, and the antibody expressed by the cells contained in the obtained culture was expressed. Analysis by FcR9_F column was performed.
培地中に添加したマンガンイオン濃度の違いによる、抗IL−6R抗体の生産性を比較した結果を図8に、抗IL−6R抗体高発現細胞数(生細胞数)の推移を図9に、抗IL−6R抗体のFcR9_Fカラムによる分析結果を図10に、抗gp130R抗体の生産性を比較した結果を図11に、抗gp130R抗体高発現細胞数(生細胞数)の推移を図12に、抗IL−6R抗体のFcR9_Fカラムによる分析結果を図13に、それぞれ示す。また図10に記載の分析結果にある各ピークの面積割合を表7に、図13に記載の分析結果にある各ピークの面積割合を表8に、それぞれ示す。 The results of comparing the productivity of the anti-IL6R antibody due to the difference in the manganese ion concentration added to the medium are shown in FIG. 8, and the transition of the number of high-expressing anti-IL-6R antibody cells (the number of viable cells) is shown in FIG. FIG. 10 shows the results of analysis of the anti-IL-6R antibody by the FcR9_F column, FIG. 11 shows the results of comparing the productivity of the anti-gp130R antibody, and FIG. 12 shows the transition of the number of high-expressing anti-gp130R antibody cells (the number of viable cells). The results of analysis of the anti-IL-6R antibody by the FcR9_F column are shown in FIG. 13, respectively. The area ratio of each peak in the analysis result shown in FIG. 10 is shown in Table 7, and the area ratio of each peak in the analysis result shown in FIG. 13 is shown in Table 8.
表7および表8より、培地としてBalanCD CHO Growth A mediumを用いたとき(実施例4)と同様、抗IL−6R抗体高発現細胞、抗gp130R抗体高発現細胞数とも、培地に0.01μMから50μMのマンガンイオンを添加して培養すると、マンガンイオン未添加時と比較し、FcR9_Fカラムとの結合力が強いピーク(ピーク3)の割合が増加していることがわかる。なお培地に50μMのマンガンイオンを添加して培養したときは、FcγRIIIa固定化分離剤との結合力がさらに弱い(すなわちADCC活性が非常に弱い抗体に相当する)ピーク(ピーク0)が確認された(図10および図13)。 From Table 7 and Table 8, as in the case of using BalanCD CHO Growth A medium as the medium (Example 4), the number of anti-IL-6R antibody high-expressing cells and anti-gp130R antibody high-expressing cell numbers was 0.01 μM to the medium. When 50 μM manganese ion was added and cultured, the ratio of peaks (peak 3) having a strong binding force with the FcR9_F column was increased as compared with the case where manganese ion was not added. When 50 μM manganese ion was added to the medium and cultured, a peak (peak 0) having a weaker binding force with the FcγRIIIa-immobilized separating agent (that is, corresponding to an antibody having very weak ADCC activity) was confirmed. (FIGS. 10 and 13).
実施例6 バイオリアクターを用いたバッチ培養におけるマンガンイオン添加の効果(その1)
(1)50μg/mLのカナマイシン、40mL/LのGlutaMAX(Thermo Fisher Scientific社製)を含んだ100mLのBalanCD CHO Growth A mediumを入れた500mLの三角フラスコ(Corning社製)に、実施例2で作製した抗IL−6R抗体高発現細胞を接種し、130rpm、37℃、8%CO2の条件下で振盪培養した。
Example 6 Effect of Manganese Ion Addition on Batch Culture Using Bioreactor (Part 1)
(1) Prepared in Example 2 in a 500 mL Erlenmeyer flask (manufactured by Corning) containing 100 mL of BalanCD CHO Growth A medium containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX (manufactured by Thermo Fisher Scientific). The anti-IL-6R antibody high-expressing cells described above were inoculated, and cultured by shaking under the conditions of 130 rpm, 37° C., and 8% CO 2 .
(2)校正したpH計、溶存酸素(DO)計をセットした2Lの滅菌済ジャーファーメンター(バイオット社製)に、50μg/mLのカナマイシン、40mL/LのGlutaMAXを含んだ900mLのBalanCD CHO Growth A mediumを入れ、(1)で培養した抗IL−6R抗体高発現細胞を0.2×106cells/mLとなるよう接種後、全量を1Lとなるよう前述の培地を追加した。前記培地には終濃度で5μMとなるよう硫酸マンガンを添加している。 (2) 900 mL of BalanCD CHO Growth containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX in a 2 L sterilized jar fermenter (manufactured by Biot) in which a calibrated pH meter and a dissolved oxygen (DO) meter are set. A medium was added thereto, and the cells highly expressing the anti-IL-6R antibody cultured in (1) were inoculated to give 0.2×10 6 cells/mL, and the above-mentioned medium was added so that the total amount became 1 L. Manganese sulfate was added to the medium to a final concentration of 5 μM.
(3)培地および細胞を入れたジャーファーメンターを制御装置(BCP:バイオット社製)にセットし、8%CO2を100mL/分で流しながら、37℃、100rpmで10日間バッチ培養した。なお培養中、pHはCO2と0.5Mの炭酸水素ナトリウム水溶液を添加することで制御し、DOは37℃での飽和溶存酸素量の50%量を保つよう制御した。培養途中で培養液を40から80mLサンプリングし、生細胞数をCountess(Thermo Fisher Scientific社製)を使用して測定し、抗体生産性を実施例2(2)に記載のELISA法(検量線作成のための標準品としてヒトIgG1(シグマ社製)を使用)にて測定した。また前記サンプリングした培養液から、実施例4(3)から(4)に記載の精製方法を用いて抗体を精製し、実施例4(5)に記載の方法でFcR9_Fカラムによる抗体分析を行なった。 (3) A jar fermenter containing a medium and cells was set in a controller (BCP: manufactured by Biot Co.), and batch culture was performed at 37° C. and 100 rpm for 10 days while flowing 8% CO 2 at 100 mL/min. During the culture, pH was controlled by adding CO 2 and 0.5 M aqueous sodium hydrogen carbonate solution, and DO was controlled so as to maintain 50% of the saturated dissolved oxygen amount at 37°C. During the culture, 40 to 80 mL of the culture solution was sampled, the number of viable cells was measured using Countess (manufactured by Thermo Fisher Scientific), and the antibody productivity was determined by the ELISA method described in Example 2 (2) (calibration curve preparation). Human IgG1 (manufactured by Sigma) was used as a standard for the measurement. The antibody was purified from the sampled culture medium using the purification method described in Example 4(3) to (4), and the antibody was analyzed by the FcR9_F column by the method described in Example 4(5). ..
比較例1
実施例6(2)で硫酸マンガンを添加しないこと、および実施例6(3)での培養期間が9日間であること以外は、実施例6と同様な方法で培養し、生細胞数および抗体生産性の測定ならびにFcR9_Fカラムによる抗体分析を行なった。
Comparative Example 1
Culture was carried out in the same manner as in Example 6 except that manganese sulfate was not added in Example 6(2), and the culture period in Example 6(3) was 9 days, and the viable cell count and antibody were used. Productivity measurements and antibody analysis with FcR9_F column were performed.
実施例6および比較例1における抗体生産性の推移を図14に、生細胞数の推移を図15に、それぞれ示す。図14および図15の結果より、培地へのマンガンイオンの添加による、抗体生産性および細胞増殖への影響はないといえる。 The changes in antibody productivity in Example 6 and Comparative Example 1 are shown in FIG. 14, and the changes in the number of viable cells are shown in FIG. 15, respectively. From the results of FIGS. 14 and 15, it can be said that the addition of manganese ions to the medium has no effect on antibody productivity and cell growth.
比較例1で得られた培養9日目の抗IL−6R抗体のFcR9_Fカラムによる分析結果および、実施例6で得られた培養10日目の抗IL−6R抗体のFcR9_Fカラムによる分析結果を図16に示す。また図16に記載の分析結果にある各ピークの面積割合を表9に示す。培地にマンガンイオンを添加して培養(実施例6)すると、マンガンイオン未添加時(比較例1)と比較し、FcR9_Fカラムとの結合力が強いピーク(ピーク3)の割合が増加していることがわかる(比較例1:27.9%[培養9日後の培養液]、実施例6:34.7%[培養10日後の培養液])。 The analysis result by the FcR9_F column of the anti-IL-6R antibody on the 9th day of culture obtained in Comparative Example 1 and the analysis result by the FcR9_F column of the anti-IL-6R antibody on the 10th day of culture obtained in Example 6 are shown. 16 shows. Table 9 shows the area ratio of each peak in the analysis result shown in FIG. When manganese ions were added to the medium and cultured (Example 6), the proportion of peaks (peak 3) having a strong binding force with the FcR9_F column was increased as compared with the case where manganese ions were not added (Comparative Example 1). (Comparative example 1: 27.9% [culture medium after 9 days of culture], Example 6: 34.7% [culture medium after 10 days of culture]).
以上の結果から、ジャーファーメンターでのバッチ培養においても、フラスコ培養のときと同様、培地にマンガンイオンを添加することで、抗体を発現可能な哺乳動物細胞が発現した抗体が有するADCC活性が向上することがわかる。 From the above results, even in batch culture with a jar fermenter, as in the case of flask culture, by adding manganese ion to the medium, the ADCC activity of the antibody expressed by the mammalian cells capable of expressing the antibody is improved. I understand that
実施例7 バイオリアクターを用いたバッチ培養におけるマンガンイオン添加の効果(その2)
(1)50μg/mLのカナマイシン、30mL/LのGlutaMAX(Thermo Fisher Scientific社製)を含んだ50mLのEX−CELL Advanced CHO Fed−batch Mediumを加えた250mLの三角フラスコ(Corning社製)に、実施例2で作製した抗IL−6R抗体高発現細胞を接種し、130rpm、37℃、8%CO2の条件下で振盪培養した。
Example 7 Effect of Manganese Ion Addition on Batch Culture Using Bioreactor (Part 2)
(1) A 250-mL Erlenmeyer flask (manufactured by Corning) to which 50 mL of EX-CELL Advanced CHO Fed-batch Medium containing 50 μg/mL of kanamycin and 30 mL/L of GlutaMAX (manufactured by Thermo Fisher Scientific) was added. The anti-IL-6R antibody high-expressing cells prepared in Example 2 were inoculated and shake-cultured under the conditions of 130 rpm, 37° C., and 8% CO 2 .
(2)校正したpH計、溶存酸素(DO)計をセットした3器の250mLのジャーファーメンター(バイオット社製)に、50μg/mLのカナマイシン、30mL/LのGlutaMAXを含んだ100mLのEX−CELL Advenced CHO Fed−batch Mediumをそれぞれ加え、(1)で培養した抗IL−6R抗体高発現細胞を0.2×106cells/mLとなるよう各リアクターに接種した後、全量を110mLとなるよう前述の培地をそれぞれ追加した。前記培地には終濃度でそれぞれ1μM、5μM、10μMとなるよう硫酸マンガンを添加している。 (2) 100 mL EX-containing 50 μg/mL kanamycin and 30 mL/L GlutaMAX in three 250 mL jar fermenters (manufactured by Biot Co.) set with a calibrated pH meter and a dissolved oxygen (DO) meter. CELL Advanced CHO Fed-batch Medium was added to each, and the cells with high anti-IL-6R antibody-expressing cells cultivated in (1) were inoculated into each reactor at 0.2×10 6 cells/mL, and the total volume was 110 mL. As mentioned above, each of the above-mentioned media was added. Manganese sulfate was added to the medium so that the final concentrations were 1 μM, 5 μM, and 10 μM, respectively.
(3)培地および細胞を加えたジャーファーメンターを制御装置(Bio Jr.8:バイオット社製)にセットし、8%CO2を100mL/分で流しながら、37℃、130rpmで10日間バッチ培養した。なお培養中、pHはCO2と0.5Mの炭酸水素ナトリウム水溶液を添加することでpH7.1を保つよう制御し、DOは37℃での飽和溶存酸素量の50%量を保つよう制御した。培養途中で培養液を1から2mLサンプリングし、生細胞数をVi−CELL XR(ベックマン・コールター社製)を使用して測定し、抗体生産性をCedex Bio(ロシュ・ダイアグノスティックス社製)を使用して測定した。また培養終了後の培養液から、実施例4(3)から(4)に記載の精製方法を用いて抗体を精製し、実施例4(5)に記載の方法でFcR9_Fカラムによる抗体分析を行なった。 (3) A jar fermenter containing a medium and cells was set in a controller (Bio Jr. 8: manufactured by Biot), and batch culture was performed at 37° C. and 130 rpm for 10 days while flowing 8% CO 2 at 100 mL/min. did. During the culture, the pH was controlled to maintain pH 7.1 by adding CO 2 and 0.5 M aqueous sodium hydrogen carbonate solution, and the DO was controlled to maintain 50% of the saturated dissolved oxygen amount at 37°C. .. During the culture, 1 to 2 mL of the culture solution was sampled, the number of viable cells was measured using Vi-CELL XR (manufactured by Beckman Coulter), and the antibody productivity was Cedex Bio (manufactured by Roche Diagnostics). Was measured using. In addition, the antibody is purified from the culture broth after the culturing using the purification method described in Example 4(3) to (4), and the antibody is analyzed by the FcR9_F column by the method described in Example 4(5). It was
比較例2
実施例7(2)で硫酸マンガンを添加しないこと以外は、実施例7と同様の方法で培養し、生細胞数および抗体生産性の測定ならびにFcR9_Fカラムによる抗体分析を行なった。
Comparative example 2
Culture was performed in the same manner as in Example 7 except that manganese sulfate was not added in Example 7(2), and the number of viable cells and antibody productivity were measured and the antibody was analyzed by the FcR9_F column.
実施例7および比較例2における抗体生産性の推移を図17に、生細胞数の推移を図18に、それぞれ示す。図17および図18の結果より、培地へのマンガンイオンの添加による、抗体生産性および細胞増殖への影響はないといえる。 The changes in antibody productivity in Example 7 and Comparative Example 2 are shown in FIG. 17, and the changes in the number of viable cells are shown in FIG. 18, respectively. From the results of FIGS. 17 and 18, it can be said that the addition of manganese ion to the medium has no effect on antibody productivity and cell growth.
比較例2および実施例7で得られた培養10日目の抗IL−6R抗体のFcR9_Fカラムによる分析結果を図19に示す。また図19に記載の分析結果にある各ピークの面積割合を表10に示す。培地にマンガンイオンを添加して培養(実施例7)すると、マンガンイオン未添加時(比較例2)と比較し、FcR9_Fカラムとの結合力が強いピーク(ピーク3)の割合が増加しており、さらにマンガンイオン濃度が高いほどピーク3の割合が増加していることがわかる(比較例2:35.9%、実施例7:36.5%(1μMマンガンイオン添加)、38.4%(5μMマンガンイオン添加)、41.0%(10μMマンガンイオン添加))。 FIG. 19 shows the results of analysis of the anti-IL-6R antibody on day 10 of culture obtained in Comparative Example 2 and Example 7 using the FcR9_F column. Table 10 shows the area ratio of each peak in the analysis result shown in FIG. When manganese ions were added to the medium and cultured (Example 7), the proportion of peaks (peak 3) having a strong binding force with the FcR9_F column increased as compared with the case where manganese ions were not added (Comparative Example 2). Further, it can be seen that the ratio of peak 3 increases as the manganese ion concentration increases (Comparative Example 2: 35.9%, Example 7: 36.5% (1 μM manganese ion added), 38.4% ( 5 μM manganese ion addition), 41.0% (10 μM manganese ion addition)).
以上の結果から、ジャーファーメンターでのバッチ培養において、培地に添加したマンガンイオンの濃度が高いほど、抗体を発現可能な哺乳動物細胞が発現した抗体が有するADCC活性が向上することがわかる。一方で、実施例4の結果(図3と図6)および実施例5の結果(図9と図12)から、より高濃度のマンガンイオン(50μM)を添加すると細胞増殖が阻害され、抗体生産量が低下することがわかる。このことから、細胞増殖を阻害しない程度の濃度(0.03μMから30μM、好ましくは0.1μMから10μM、より好ましくは1μMから10μM)のマンガンイオンを添加すれば、抗体産生量を低下させずに高いADCC活性を有する抗体を発現できるといえる。 From the above results, it can be seen that in batch culture with a jar fermenter, the higher the concentration of manganese ion added to the medium, the more the ADCC activity possessed by the antibody expressed by the mammalian cells capable of expressing the antibody. On the other hand, from the results of Example 4 (FIGS. 3 and 6) and the results of Example 5 (FIGS. 9 and 12), addition of a higher concentration of manganese ion (50 μM) inhibits cell growth and antibody production. It can be seen that the amount decreases. From this, if manganese ion is added at a concentration that does not inhibit cell growth (0.03 μM to 30 μM, preferably 0.1 μM to 10 μM, more preferably 1 μM to 10 μM), the amount of antibody produced does not decrease. It can be said that an antibody having high ADCC activity can be expressed.
Claims (5)
前記培養工程を0.01μMから50μMのマンガンイオンを添加した培地で行なうことで、抗体依存性細胞傷害活性が向上した抗体を製造する方法。 In the method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody, and a step of collecting the antibody expressed by the mammalian cell contained in the obtained culture,
A method for producing an antibody having improved antibody-dependent cellular cytotoxicity by performing the culturing step in a medium supplemented with 0.01 to 50 µM manganese ion.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2018243922 | 2018-12-27 | ||
JP2018243922 | 2018-12-27 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2020103280A true JP2020103280A (en) | 2020-07-09 |
JP7469593B2 JP7469593B2 (en) | 2024-04-17 |
Family
ID=71449805
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2019224246A Active JP7469593B2 (en) | 2018-12-27 | 2019-12-12 | Method for producing an antibody having improved antibody-dependent cellular cytotoxicity |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP7469593B2 (en) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PE20160218A1 (en) | 2013-07-23 | 2016-05-09 | Biocon Ltd | METHODS TO CONTROL LEVELS OF FUCOSILATION IN PROTEINS |
-
2019
- 2019-12-12 JP JP2019224246A patent/JP7469593B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
JP7469593B2 (en) | 2024-04-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10508154B2 (en) | Process and methods for efficient manufacturing of highly pure asymmetric antibodies in mammalian cells | |
JP7083802B2 (en) | How to prepare glycosylated immunoglobulin | |
US11267899B2 (en) | Afucosylated protein, cell expressing said protein and associated methods | |
TWI513818B (en) | Fucosylation-deficient cells | |
WO2012041768A1 (en) | Production of low fucose antibodies in h4-ii-e rat cells | |
JP6351598B2 (en) | Compositions and methods for producing glycoproteins | |
SG173796A1 (en) | Metabolic engineering of a galactose assimilation pathway in the glycoengineered yeast pichia pastoris | |
KR20100065332A (en) | Production method | |
JP2019088305A (en) | Overexpression of n-glycosylation pathway regulators for modulating glycosylation of recombinant proteins | |
TWI781973B (en) | Method for in vitro glycoengineering of antibodies | |
JP2020188737A (en) | Production method of antibody with improved antibody dependent cell mediated cytotoxicity activity | |
JP7469593B2 (en) | Method for producing an antibody having improved antibody-dependent cellular cytotoxicity | |
JP2022094382A (en) | Method for producing antibody having improved antibody-dependent cellular cytotoxicity | |
WO2014126254A1 (en) | Membrane-binding protein expression vector | |
JP7081139B2 (en) | New promoter and expression vector containing it | |
JP5512514B2 (en) | Heavy chain variants resulting in improved immunoglobulin production | |
JP2023522417A (en) | Enzyme and pathway regulation using sulfhydryl compounds and their derivatives | |
Jung | Engineering mammalian cell line for N-linked glycosylation control | |
WO2023180374A1 (en) | New glutamine synthetase variants as selection marker | |
WO2023180398A1 (en) | Bacterial glutamine synthetase as selection marker in mammalian cells | |
AU2018203733A1 (en) | Method for the production of a glycosylated immunoglobulin | |
Bennun et al. | 4 Engineering of the Product |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200302 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20221116 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20231020 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20231031 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231218 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20240305 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20240318 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7469593 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |