JP6486310B2 - アルファ2,3−およびアルファ2,6−シアリル化を含む組換えfsh - Google Patents
アルファ2,3−およびアルファ2,6−シアリル化を含む組換えfsh Download PDFInfo
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- JP6486310B2 JP6486310B2 JP2016198588A JP2016198588A JP6486310B2 JP 6486310 B2 JP6486310 B2 JP 6486310B2 JP 2016198588 A JP2016198588 A JP 2016198588A JP 2016198588 A JP2016198588 A JP 2016198588A JP 6486310 B2 JP6486310 B2 JP 6486310B2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
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- C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
- C12Y204/99006—N-Acetyllactosaminide alpha-2,3-sialyltransferase (2.4.99.6)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Description
ヒトFSH
FSHアルファポリペプチドの遺伝子のコード領域を、FiddesおよびGoodman(1981年)に従って使用した。この配列は、AH007338として寄託されており、構築時には、このタンパク質配列の他の変異体は存在しなかった。この配列は、本明細書中では配列番号1として参照される。
α2,3−シアリルトランスフェラーゼ:ベータガラクトシドアルファ−2,3−シアリルトランスフェラーゼ4(α2,3−シアリルトランスフェラーゼ、ST3GAL4)の遺伝子のコード領域を、KitagawaおよびPaulson(1994年)に従って使用した。この配列は、L23767として寄託されており、本明細書中では配列番号3として参照される。
FSH発現ベクターの構築
FSHアルファポリペプチド(AH007338、配列番号1)のコード配列、およびFSHベータポリペプチド(NM_003032、配列番号2)を、プライマーの組合せFSHa−fwおよびFSHa−revならびにFSHb−fwおよびFSHb−recをそれぞれ使用して、PCRにより増幅した。
FSHa-fw 5'-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3'
FSHa-rev 5'-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3'
FSHb-fw 5'-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3'
FSHb-rev 5'-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-3'
ST3発現ベクターの構築
ベータガラクトシドアルファ−2,3−シアリルトランスフェラーゼ4(ST3、L23767、配列番号3)のコード配列を、プライマーの組合せ2,3STfwおよび2,3STrevを使用してPCRにより増幅した。
2,3STfw 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3'
2,3STrev 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3'
ST6発現ベクターの構築
ベータガラクトサミドアルファ−2,6−シアリルトランスフェラーゼ1(ST6、NM_003032、配列番号4)のコード配列を、プライマーの組合せ2,6STfwおよび2,6STrevを使用してPCRにより増幅した。
2,6STfw 5'-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3'
2,6STrev 5'-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3'
PER.C6細胞におけるpFSH A+Bの安定的発現。クローンのトランスフェクション単離およびスクリーニング。
FSHを産生するPer.C6クローンを、単一プラスミドからFSHの両ポリペプチド鎖を発現させることにより産生した(実施例1を参照)。
シアリル化のレベルは、α2,3−またはα2,6−シアリルトランスフェラーゼを過剰発現する細胞で増加する。PER.C6細胞を発現するFSHにおけるpST3またはpST6の安定的な発現;クローンのトランスフェクション単離およびスクリーニング。
FSHの両ポリペプチド鎖を既に発現しているPer.C6細胞においてα2,3シアリルトランスフェラーゼまたはα2,6シアリルトランスフェラーゼを別々のプラスミド(実施例2および3を参照)から発現させることにより、高度にシアリル化されたFSHを産生するPer.C6クローンを生成した(実施例4を参照)。実施例4で示したようなPER.C6(登録商標)細胞から生成した4つのクローンを、生産性、良好な増殖特性、機能的タンパク質の産生、および産生されたFSH(幾らかのシアリル化を含む)などの特徴によって選択した。
Per.C6が産生したFSHアイソフォームのpIの等電点電気泳動による分析。
電気泳動は、電場による荷電された分子の溶媒中の移動と定義される。電場中の生体分子の移動度は、電場の強さ、分子の正味電荷、分子のサイズおよび形状、分子が移動する媒体のイオン強度および特性に依存するだろう。
Per.C6 FSHのシアル酸結合の分析
レクチンに基づくグリカン識別法を使用して、複合糖質を分析した。この方法では、ニトロセルロースに結合した糖タンパク質および複合糖質を特徴づけることができる。レクチンは、特定の部分、例えばα2,3結合シアル酸を選択的に認識する。添加されたレクチンは、ステロイドハプテンジゴキシゲニンと結合し、これによって結合したレクチンの免疫学的検出が可能になる。
総シアル酸の定量
シアル酸は、単糖類と考えられているタンパク質結合炭水化物であり、ガラクトース、マンノース、グルコサミン、ガラクトサミン、およびフコースのような他のモノサッカライドとの組合せで生じる。
α2,3、α2,6、およびα2,8シアル酸の相対量の定量
精製rFSH(実施例11)のα2,3、α2,6、およびα2,8シアル酸の相対パーセント量を、公知の技術を使用して測定した。
モノ、ジ、トリ、およびテトラアンテナシアリル化構造の相対量の定量
精製rFSH(実施例11)から抽出したグリカンにあるモノ、ジ、トリ、およびテトラシアリル化構造の相対パーセント量を、公知の技術を使用して測定した。
rFSHの代謝クリアランス率の測定
Per.C6 FSH試料の代謝クリアランス率(MCR)を測定するために、意識のある雌ラット(1クローン当たり3匹の動物)の尾部静脈に、rFSHのボーラスを時点ゼロで注射した(1〜10μg/ラット、試料のELISA定量に基づいて、DRG EIA 1288)。血液試料(400μl)を、試験試料注射の1、2、4、8、12、24、および32時間後に尾部先端から採取した。血清を遠心分離により収集し、ELISA(DRG EIA 1288)によりFSH含有量についてアッセイした。
Steelman−Pohley in vivoアッセイ
FSHのシアル酸含有量の増加が、その結果として生物学的効果の増加をもたらすことを示すために、高度にシアリル化されたFSH、例えば実施例5で産生されたものなど、によるラット卵巣の重量増加を検査した。
産生および精製の概要
無血清培地に懸濁培養したPER.C6細胞中でFSHを産生するための手順を開発した。その手順は下記に記載されており、数個のFSH産生PER.C6細胞系に適用した。
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Grundmann,U., Nerlich,C., Rein,T. and Zettlmeissl, G. (1990). Complete cDNA sequence encoding human beta-galactoside alpha-2,6-sialyltransferase. G Nucleic Acids Res. 18 (3), 667
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Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO and Vutyavanich T. (1987). Structure-function relationships of gonadotropins. Recent Prog Horm Res.;43,:383-429.
Saxena BB and Rathnam P. (1976) Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands. J Biol Chem. 251(4), 993-1005
Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 53(6), 604-616.
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卵胞刺激ホルモンアルファポリペプチド
受託番号AH007338
FSHアルファのヌクレオチド配列
1 ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT
61 GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA
121 TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA
181 TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG
241 TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG
301 GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA A
FSHアルファのタンパク質配列
1 MKTLQFFFLF CCWKAICCNS CELTNITIAI EKEECRFCIS INTTWCAGYC YTRDLVYKDP
61 ARPKIQKTCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS
121 YCSFGEMKE
配列番号2
卵胞刺激ホルモンベータポリペプチド
受託番号NM_000510
FSHベータのヌクレオチド配列
1 ATGAAGACAC TCCAGTTTTT CTTCCTTTTC TGTTGCTGGA AAGCAATCTG CTGCAATAGC
61 TGTGAGCTGA CCAACATCAC CATTGCAATA GAGAAAGAAG AATGTCGTTT CTGCATAAGC
121 ATCAACACCA CTTGGTGTGC TGGCTACTGC TACACCAGGG ATCTGGTGTA TAAGGACCCA
181 GCCAGGCCCA AAATCCAGAA AACATGTACC TTCAAGGAAC TGGTATATGA AACAGTGAGA
241 GTGCCCGGCT GTGCTCACCA TGCAGATTCC TTGTATACAT ACCCAGTGGC CACCCAGTGT
301 CACTGTGGCA AGTGTGACAG CGACAGCACT GATTGTACTG TGCGAGGCCT GGGGCCCAGC
361 TACTGCTCCT TTGGTGAAAT GAAAGAATAA
FSHベータのタンパク質配列
1 MKTLQFFFLF CCWKAICCNS CELTNITIAI EKEECRFCIS INTTWCAGYC YTRDLVYKDP
61 ARPKIQKTCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS
121 YCSFGEMKE
配列番号3
ベータガラクトシドアルファ−2,3−シアリルトランスフェラーゼ4
受託番号L23767
ST3GAL4のヌクレオチド配列
1 ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG
61 TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG
121 AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC
181 TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA
241 TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA
301 GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG
361 GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC
421 GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC
481 AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC
541 AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG
601 ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC
661 TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT
721 GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG
781 GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT
841 GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC
901 AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG
961 CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA
ST3GAL4のタンパク質配列
1 MCPAGWKLLA MLALVLVVMV WYSISREDRY IELFYFPIPE KKEPCLQGEA ESKASKLFGN
61 YSRDQPIFLR LEDYFWVKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV
121 VVGNGHRLRN SSLGDAINKY DVVIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN
181 NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA
241 DKLLSLPMQQ PRKIKQKPTT GLLAITLALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL
301 KSMAGSGHNV SQEALAIKRM LEMGAIKNLT SF
配列番号4
ベータガラクトサミドアルファ−2,6シアリルトランスフェラーゼ1
受託番号NM_003032
ST6GAL1のヌクレオチド配列
1 ATGATTCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT
61 GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG
121 CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC
181 CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT
241 CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC
301 TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC
361 AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG
421 CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC
481 AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT
541 TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA
601 GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA
661 CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG
721 AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGGAATCC TAATTGTATG GGACCCATCT
781 GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC
841 TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG
901 CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC
961 CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT
1021 TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC
1081 GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA TTTGGTGAAG
1141 CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC
1201 TTCCGGACCA TTCACTGCTA A
0p−ST6GAL1のタンパク質配列
1 MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS
61 QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN
121 KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP
181 WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE
241 KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM
301 PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF
361 DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC
Claims (10)
- α2,3−およびα2,6−シアリル化を有し、かつ、総シアリル化の10%〜90%がα2,3−シアリル化である組換えFSH(rFSH)の製造方法であって、
ヒト細胞株においてrFSHを産生または発現させる工程を含み、ここでヒト細胞株はα2,3−シアリルトランスフェラーゼ遺伝子を導入することによって改変されている、方法。 - ヒト細胞株は、内因性のα2,6−シアリルトランスフェラーゼ活性を有する、請求項1に記載の方法。
- ヒト細胞株は、Per.C6細胞株である、請求項1または2に記載の方法。
- Per.C6細胞株は、内因性シアリルトランスフェラーゼ活性を有する、請求項3に記載の方法。
- 請求項1〜4のいずれか一項に記載の方法により製造された組換えFSH(rFSH)。
- 組換えFSH(rFSH)を生産する方法であって、Per.C6細胞株において、rFSHを産生または発現させる工程を含み、Per.C6細胞株はα2,3−シアリルトランスフェラーゼ遺伝子を導入することによって改変されている、方法。
- 請求項6に記載の方法により製造された組換えFSH(rFSH)。
- 請求項5または7に記載の組換えFSHを含む、医薬組成物。
- hCGおよび/またはLHをさらに含む、請求項8に記載の医薬組成物。
- 不妊の処置に用いるための、請求項8または9に記載の医薬組成物。
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