JP6060167B2 - 線維芽細胞増殖因子21変異体 - Google Patents
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Classifications
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- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
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- 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
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
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- A61P3/00—Drugs for disorders of the metabolism
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Chemical Kinetics & Catalysis (AREA)
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- Child & Adolescent Psychology (AREA)
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- Emergency Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Description
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX1LLEDGYNVYQSEAHGLX2LHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPX3X4LVX5PSQLLSPSFLG(配列番号13)
であり、ここで、X1はLまたはDであり、X2はPまたはWであり、X3はLまたはYであり、X4はSまたはRであり、X5はGまたはEである、変異体を提供する。
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG(配列番号1)
である、変異体を提供する。
CHOK1SV細胞におけるFGF21変異体の発現
チャイニーズハムスター卵巣(CHOK1SV)細胞を使用して哺乳動物細胞発現系において本発明のFGF21変異体を産生する。FGF21変異体をコードする遺伝子を、グルタミンシンテターゼ(GS)を含有する発現プラスミド骨格(pEE12.4ベースのプラスミド)内でサブクローニングする。FGF21変異体をコードするcDNA配列を、組織培養培地内への所望の産物の分泌を増強するために好ましいシグナルペプチド配列のコード配列とフレーム内で融合する。好ましいシグナルペプチド配列は、配列番号3、配列番号4、配列番号5、または配列番号6のアミノ酸配列に示されるようなポリペプチドである。
3T3−L1−βKlotho線維芽細胞グルコース摂取アッセイ
3T3−L1−βKlotho線維芽細胞は、野生型マウスβKlothoのコード配列およびブラストサイジン耐性マーカーを含有するCMVにより駆動される哺乳動物発現ベクターのレトロウイルス形質導入により3T3−L1線維芽細胞から生成する。15μMのブラストサイジンの存在下で14日間の増殖後にブラストサイジン耐性細胞を選択し、βKlothoタンパク質発現を、抗βKlotho抗体を用いた免疫ブロットにより検証する。実験での使用のために播種するまで、10%のウシ血清、および15μMのブラストサイジンを有するダルベッコ改変イーグル培地(DMEM)中に3T3−L1−βKlotho線維芽細胞を維持する。
ヒト293細胞−βKlotho−SREルシフェラーゼアッセイ
293−βKlotho−SRElucレポーター細胞の構築:
37℃、5%CO2にて、ダルベッコ改変イーグル培地中に10%ウシ胎児血清(FBS)を含有する増殖培地中でHEK−293(ヒト胎児腎臓細胞)を培養する。CMVプロモーターにより駆動されるβKlotho発現カセットを含有するプラスミドおよび血清反応要素(SRE)により駆動されるリスフェラーゼ発現カセットを含有するプラスミドを用いて細胞を共トランスフェクトする。βKlotho発現プラスミドもまた、アミノグリコシド系抗生物質G418に対する耐性を与えるためにSV40プロモーターにより駆動されるネオマイシンホスホトランスフェラーゼ発現カセットを含有する。トランスフェクトプラスミドがゲノム内に組み込まれている細胞を選択するために600μg/mLのG418を用いてトランスフェクトしたHEK−293細胞を選択する。希釈により選択した細胞をクローニングし、FGF21の添加の24時間後にルシフェラーゼ産物の増加について試験する。最大のFGF21に依存したルシフェラーゼの増加を示しているクローンを、関連するFGF21変異体活性を測定するために使用する細胞株として選択する。
293−βKlotho−SREluc細胞をリンスし、CD293懸濁培養培地(Invitrogen)内に入れる。37℃、6%CO2、125rpmにて細胞を懸濁液中で一晩増殖させる。細胞を計数し、遠心分離によりペレット化し、0.1%BSAを含有するCD293培地中で再懸濁する。25,000個の細胞/ウェルにて細胞を白色96ウェルプレート内に入れる。CD293/0.1%BSA中での4倍連続希釈を、100nM〜0.006nMの最終濃度で8個の希釈物を生成するために各FGF21変異体について調製する。希釈物を3連で細胞に加え、37℃、5%CO2にて16〜20時間インキュベートする。等体積のOneGlo(商標)ルシフェラーゼ基質(Promega)を添加し、相対蛍光を測定することによりルシフェラーゼレベルを決定する。曲線を適合するために4パラメータロジスティックモデル(XLfitバージョン5.1)を使用してデータを分析し、EC50を決定する。
哺乳動物細胞における製造の間のチロシン硫酸化
ヒト野生型FGF21は、CHOK1SV細胞における哺乳動物タンパク質発現の間、179位におけるチロシン硫酸化に影響を受けやすい(データは示さず)。この硫酸化は不均質を生じることを導き、タンパク質の異なる形態(すなわち硫酸化を有するおよび有さない)が発生し得ることを意味する。生成物の同質性は生物製剤の所望の特性である。治療タンパク質の産生の間に起こる翻訳後修飾は望ましくない。なぜなら、修飾は活性または他の生物薬剤特性の相違を導き得るからである。
P115Wは凝集を促進するのに対して、L98Dは物理的安定性および製剤中のベンジルアルコールとの適合性を増加させる
タンパク質自己会合および凝集の量を測定するために、分析的サイズ排除クロマトグラフィー(SEC)法を使用して、高分子量凝集体のパーセント(%HMW)を測定する。タンパク質の初期ストック溶液をSECにより特徴付けて、HMWの開始レベルを決定する(表3)。
インビボでのタンパク質分解
オスのカニクイザル、n=2/群に、配列番号1のFGF21変異体の単回の2mg/kg注射を皮下投与する。活性化合物の量を定量化するために質量分析によりインビボタンパク質分解の24時間の評価の間、血清を経時的に得る(0.25〜12時間後に引き抜く)。
Ob/obマウスモデルにおけるグルコース低下
オスのob/ob(B6.V−Lepob/Lep+/OlaHsd)マウスおよび年齢を一致させたob/m痩せた対照(B6.V−Lep+/OlaHsd)は、到着時に7〜8週齢で、治療開始時に10〜11週齢である。到着時に全てのマウスをケージ当たり3匹で収容し、治療の開始前に3週間馴れさせる。マウスにPurina Rodent Chow5015を与え、水を自由に与える。マウスを70°Fに設定した周囲温度で12時間明/暗周期に収容する。治療の開始の前日に、マウスを2時間絶食させ、尾出血によりヘパリン化毛細管内に血液試料を採取する。Ascensia Contor血糖測定器を用いて血糖値を測定し、Meso Scaleマウス/ラットインスリンアッセイキット(Meso Scale Discovery、Gaithersburg、MD)を使用して血漿インスリンレベルを定量する。治療の開始日(0日)に、前日の体重、血糖、および血漿インスリンに基づいてマウスを分類する。無菌食塩水(0.9%NaCl)を用いてFGF21変異体を希釈し、ミニ浸透圧Alzetポンプにより皮下投与する。5日目に、明サイクルの開始後約2時間で、供給した血糖および血漿インスリンレベルを測定する。5日目に全てのマウスを一晩絶食させ、6日目に経口ブドウ糖負荷試験(OGTT)を実施する。グルコース(2g/kg)の経口投与前に尾を切断することによりヘパリン化した毛細管内にマウスを出血させる。経口グルコース投与後、30、60および120分にさらに血液試料を採取する。Cayman Chemicals製のグルコースアッセイキットを用いて血漿グルコースを測定する。4パラメータロジスティック回帰モデルフィットを6日目に、正規化したグルコースAUC値で実施する。
配列番号1−FGF21変異体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG
配列番号2−野生型FGF21(ホモサピエンス)
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
配列番号3−ヒトトランスフェリン(hTrf)シグナルペプチド
MRLAVGALLVCAVLGLCLA
配列番号4−ヒト線維芽細胞増殖因子結合タンパク質−1(hFGFP−1)シグナルペプチド
MKICSLTLLSFLLLAAQVLLVEG
配列番号5−ウシリゾチームシグナルペプチド
MKALVILGFLFLSVAVQG
配列番号6−マウス軽鎖(mカッパ)シグナルペプチド
METDTLLLWVLLLWVPGSTG
配列番号7−FGF21変異体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVGPSQLLSPSFLG
配列番号8−FGF21変異体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLWLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPYSLVEPSQLLSPSFLG
配列番号9−FGF21変異体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPYSLVEPSQLLSPSFLG
配列番号10−FGF21変異体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLWLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG
配列番号11−FGF21変異体
DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS
配列番号12−(DNA)FGF21変異体
CACCCTATCCCTGACTCCAGCCCTCTGCTGCAGTTTGGCGGACAGGTCCGGCAGCGGTACCTGTACACCGACGACGCCCAGCAGACCGAGTGCCACCTGGAAATCCGGGAGGACGGCACCGTGGGCTGTGCCGCCGACCAGTCCCCTGAGTCCCTGCTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCTCCCGGTTCCTGTGCCAGAGGCCTGATGGCGCCCTGTACGGCTCCCTGCACTTCGACCCTGAGGCCTGCTCCTTCCGGGAGGACCTGCTGGAAGATGGCTACAACGTGTACCAGTCCGAGGCTCACGGCCTGCCTCTGCATCTGCCTGGCGACAAGTCCCCCCACCGGAAGCCTGCTCCTAGGGGCCCTGCCAGATTCCTGCCACTGCCTGGCCTGCCTCCAGCTCTGCCTGAGCCTCCTGGCATCCTGGCCCCTCAGCCTCCAGACGTGGGCTCCTCCGACCCTCTGCGGCTGGTCGAGCCTTCCCAGCTGCTGAGCCCTAGCTTCCTGGGC
配列番号13−FGF21変異体−コンセンサス
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX1LLEDGYNVYQSEAHGLX2LHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPX3X4LVX5PSQLLSPSFLG
X1はLまたはDであり、
X2はPまたはWであり、
X3はLまたはYであり、
X4はSまたはRであり、
X5はGまたはEである。
Claims (3)
- アミノ酸配列が、
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG(配列番号1)
である、ヒト線維芽細胞増殖因子21(FGF21)の変異体。 - 2型糖尿病、肥満、脂質異常症、もしくはメタボリックシンドローム、またはそれらの任意の組み合わせを治療するための、請求項1に記載の変異体と、少なくとも1種の薬学的に許容可能な担体、希釈剤、または賦形剤とを含む医薬組成物。
- 2型糖尿病、肥満、脂質異常症、もしくはメタボリックシンドローム、あるいはそれらの任意の組み合わせの治療に使用する医薬を製造するための、請求項1に記載の変異体の使用。
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US201161542906P | 2011-10-04 | 2011-10-04 | |
US61/542,906 | 2011-10-04 | ||
PCT/US2012/057053 WO2013052311A1 (en) | 2011-10-04 | 2012-09-25 | Fibroblast growth factor 21 variants |
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