JP2008073057A - 複数のctp付加を有するホルモンアナログ - Google Patents
複数のctp付加を有するホルモンアナログ Download PDFInfo
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- JP2008073057A JP2008073057A JP2007306928A JP2007306928A JP2008073057A JP 2008073057 A JP2008073057 A JP 2008073057A JP 2007306928 A JP2007306928 A JP 2007306928A JP 2007306928 A JP2007306928 A JP 2007306928A JP 2008073057 A JP2008073057 A JP 2008073057A
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Abstract
【解決手段】本発明の修飾ペプチド及びタンパク質はC端にヒト絨毛性性腺刺激ホルモンのカルボキシル端部位を含む、少なくとも2つの直列付加を含む。これらの”CTPユニット”は本質的に、HCG−β配列の112−118位から145位に本来存在するアミノ酸配列またはそれらの保存的修飾から構成される。
【選択図】なし
Description
本発明はin vivoにおける安定性が向上するように修飾を受けた医薬用化合物に関する。より具体的には本発明はヒト絨毛性血清性性腺刺激ホルモンのカルボキシル端ペプチドを直列に付加すること(tandem extension)による、薬学的に重要なペプチドの修飾に関する。
1990年9月7日に公開され、本明細書中に参考文献として取り込まれるPCT出願第WO90/09800号は、生殖ホルモンの様々な修飾について記載している。更に、この公開は、タンパク薬剤及び一般のホルモンのin vivoにおける生物学的半減期を延長するために、HCG−βサブユニットまたはその変異体のカルボキシル端部位を、該タンパク薬剤等のカルボキシル端に結合するという修飾を行うことができることを開示する。PCT出願の開示は多数のHCG−β鎖カルボキシル端部位(CTB)を直列に付加することを特異的に記載するものではない。本発明はそのような直列の付加を記載する。
本発明はカルボキシル端に少なくとも2つのCTP配列の直列付加を含むことにより生物学的半減期が延長した、修飾ペプチド及びタンパク質を提供する。これらの付加タンパク質は未修飾のものと同等の生物学的機能を有するが、生物学的半減期の延長により投与量を減らすことができるなどの利点を有する。
生物学的に重要なペプチドまたはタンパク質はすべて本発明の方法に従った修飾を受け得る。従って、このような修飾の対象候補に含まれるのは上記の4つのヒト”生殖”ホルモン、特にそれらのβ鎖;インスリン;ヒト成長ホルモン;エンケファリン;ACTH;グルカゴン;等のようなペプチドホルモンである。また本発明の修飾を受けるものとして有用なものにはインスリン様成長因子;表皮成長因子;酸性及び塩基性繊維芽細胞成長因子;血小板由来成長因子などの様々な成長因子、顆粒球CSF;大食細胞CSF等のような様々なコロニー刺激因子、並びにIL−2,IL−3及びそのほか非常に多くのインターロイキンタンパク質;様々なインターフェロン;腫瘍壊死因子;等のような様々なサイトカインがある。また本発明の方法を用いる候補として、黄体形成ホルモン放出ホルモン(LHRH);ソマトスタチン;成長ホルモン放出因子(GHRF);及びエンドルフィンのような短いペプチド配列がある。肺胞界面活性タンパク質;ナトリウム利尿性因子;アドヘシン;受容体ペプチド;一般の受容体結合リガンド;抗体及びその断片;並びに、所望の生物学的機能を有するその他の有用なペプチドまたはタンパク質、等のタンパク質薬剤もまた本明細書中に記載された方法に従って修飾を受け得る。
酸性:生理的pHにおいて残基はHイオンを失って負の電荷をもち、水性溶液に誘引される。このため、この残基を含むペプチドが生理的pHの水性環境中に存在する場合、残基はペプチドの構造中、表面に位置しようとする。
酸性:アスパラギン酸及びグルタミン酸
塩基性/非環式:アルギニン、リジン
塩基性/環式:ヒスチジン
中性/極性/小型:グリシン、セリン、システイン
中性/無極性/小型:アラニン
中性/極性/大型/非芳香族:スレオニン、アスパラギン、グルタミン
中性/極性/大型/芳香族:チロシン
中性/無極性/大型/非芳香族:バリン、イソロイシン、ロイシン、メチオニ ン
中性/無極性/大型/芳香族:フェニルアラニン、及びトリプトファン
遺伝子にコードされている第2アミノ酸のプロリンは専門的には中性/無極性/大型/環式及び非芳香族のグループに入るが、ペプチド鎖の2次構造への既知の効果のため特別なケースとなり、従って、この定義グループには含まれない。 もし本発明の修飾ペプチドが遺伝子の修飾によって構築される場合、CTPユニットは遺伝子にコードされるアミノ酸による置換のみ含むことになるが、CTPユニットが標準的なペプチド合成法、例えば固相ペプチド合成法によって合成され、そして受け手のペプチドまたはタンパク質のC端に、例えば酵素的に結合される場合、アミノイソブチル酸(Aib)、フェニルグリシン(Phg)等のような遺伝子にコードされないアミノ酸も類似の相似物と置換され得る。
Sar及びbeta−Ala及びAibは中性/無極性/小型;
t−BuA、t−BuG、N−MeIle、Nle、Mvl及びChaは中性/無極性/大型/非芳香族;
Ornは塩基性/非環式;
Cyaは酸性;
Cit、アセチルLys及びMSOは中性/極性/大型/非芳香族、そして
Phg、Nal、Thi及びTicは中性/無極性/大型/芳香族。
当該技術分野において一般に知られているように、本発明の修飾ペプチド及びタンパク質は目的の応用に従って、ラベル、薬剤、標的因子、担体、固相支持体等に結合していてもよい。ラベルされた型の修飾生化学薬剤は代謝の結果を追跡するために使用されても良いし;この目的に適したラベルには特にヨウ素131、テクネチウム99、インジウム111等のようなラジオアイソトープラベルが含まれる。ラベルはまた、分析系において修飾タンパク質またはペプチドの検出を媒介するのに使用されてもよい;この場合には、酵素ラベル、蛍光ラベル、色原体ラベル等と同様に、ラジオアイソトープを使用することもできる。このようなラベルの使用はペプチドまたはタンパク質がそれ自身、抗体または受容体リガンドのような標的因子である場合、特に有用である。
本発明は、好ましい態様として以下のものを含む。
1.2つ若しくはそれ以上の直列CTPユニットをC端に付加された、生物学的活性を有するペプチドまたはタンパク質を含む、修飾ペプチドまたはタンパク質。
5.修飾ペプチドまたはタンパク質をコードし、発現をもたらすための制御配列に機能的に結合されているDNAを含み;
組み換え宿主細胞内に含まれる場合に該修飾ペプチドまたはタンパク質を生産できる、組み換え発現システムであって、
該修飾ペプチドまたはタンパク質は、2つ若しくはそれ以上の直列CTPユニットをC端に付加された、生物学的活性を有するペプチドまたはタンパク質を含む、
組み換え発現系システム。
7.2つ若しくはそれ以上の直列CTPユニットをC端に付加された、生物学的活性を有するペプチドまたはタンパク質を含む修飾ペプチドまたはタンパク質の製造方法であって、
該コーディングDNAが発現される条件下で態様6の請求の細胞を培養し;そして
該修飾ペプチドまたはタンパク質を回収する、
ことからなる方法。
10.追加の置換体が固相支持体、ラベル、薬剤、糖質、及び免疫原性を与える担体から選ばれる、態様9に記載の修飾ペプチドまたはタンパク質。
11.態様1に記載の修飾ペプチドまたはタンパク質と特異的に免疫反応性をもつ抗体。
実施例1
2つのCTPユニット直列付加を有するヒトβサブユニットの分離
図1A及び1BはヒトFSHのβ鎖が2つのCTPユニットを含むように修飾されている発現ベクターのコンストラクションを示す。図1Bに示されるように、より付加されたβサブユニットを得るために、1つのCTPユニットが付加したヒトFSHβ鎖の3’端のHindIII部位をヒトHCG−β遺伝子の3’端由来のCTPユニットを結合するのに用いる。次に付加された型のFSH−β鎖の、ホニュウ類の細胞内での生産を可能にする発現システムを得るため、hFSH−β(CTP)2遺伝子を発現ベクターpM2へ結合する。宿主発現ベクターのコンストラクションはMatzuk,M.M.et al.,Proc Natl Acad Sci USA (1987) 84:6354−6358;Matzuk,M.M.et al.,J Cell Biol(1988) 106:1049−1059に記載されている。
実施例2
CTP直列付加の効果
上記の実施例1で示したように作成した、2つのCTPユニットを付加したβサブユニットを含むヒトFSHをラットに投与した。24匹のSprague−Dawleyメスのラットをこの実験に使用した。12のラットにはそれぞれ、MEM培地中で作成された10 IUの未修飾FSHを投与し、残りの12のラットにはそれぞれ、MEM培地中で作成された10 IUのhFSHβ(CTP)2付加FSHを投与した。血清を、投与直後、及び1時間以内に数回、そして2、4、及び8時間後に採取した。FSHホルモンに対する標準的なラジオイムノアッセイを用いて血清を解析した。結果を図2に示す。
Claims (7)
- 2つ若しくはそれより多くの直列CTPユニットをC末端に付加された、生物学的活性を有するペプチドまたはタンパク質を含む、修飾ペプチドまたはタンパク質であって、
前記CTPユニットは、ヒト絨毛性性腺刺激ホルモンのβサブユニットの112−118位から145位に天然に存在するアミノ酸配列、
非修飾型の生物学的活性を保持している、前記修飾ペプチドまたはタンパク質。 - 請求項1の修飾ペプチドまたはタンパク質をコードするDNA。
- 修飾ペプチドまたはタンパク質をコードし、発現をもたらすための制御配列に機能的に結合されているDNAを含み;
組換え宿主細胞内含まれる場合に該修飾ペプチドまたはタンパク質を生産できる、組換え発現システムであって、
該修飾ペプチドまたはタンパク質は、2つ若しくはそれより多くの直列CTPユニットをC末端に付加された、生物学的活性を有するペプチドまたはタンパク質を含み、非修飾型の生物学的活性を保持している、
ここにおいて、前記CTPユニットは、ヒト絨毛性性腺刺激ホルモンのβサブユニットの112−118位から145位に天然に存在するアミノ酸配列からなる、
前記組換え発現システム。 - 請求項3の発現システムで形質導入または形質転換された、組換え宿主細胞。
- 2つ若しくはそれより多くの直列CTPユニットをC末端に付加された、生物学的活性を有するペプチドまたはタンパク質を含む、修飾ペプチドまたはタンパク質であって、
前記CTPユニットは、ヒト絨毛性性腺刺激ホルモンのβサブユニットの112−118位から145位に天然に存在するアミノ酸配列からなる、
非修飾型の生物学的活性を保持している、前記修飾ペプチドまたはタンパク質、の製造方法であって、
前記修飾ペプチドまたはタンパク質が発現される条件下で請求項4記載の細胞を培養し;そして
前記修飾ペプチドまたはタンパク質を回収する
ことを含む、前記方法。 - 生物学的活性を有するペプチドまたはタンパク質の生物学的半減期を延長する方法であって、該ペプチドまたはタンパク質に少なくとも2つの直列CTPユニットを付加する
ここにおいて、前記CTPユニットは、ヒト絨毛性性腺刺激ホルモンのβサブユニットの112−118位から145位に天然に存在するアミノ酸配列からなる、
ことを含む、前記方法。 - さらに、固相支持体、ラベル、薬剤、糖質、免疫原性を与える単体、及びリン酸化残基からなるグループから選択される追加の置換体と結合している、請求項1に記載の修飾ペプチドまたはタンパク質。
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US07/771,262 US6225449B1 (en) | 1991-10-04 | 1991-10-04 | Hormone analogs with multiple CTP extensions |
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JP50709393A Expired - Lifetime JP4081137B2 (ja) | 1991-10-04 | 1992-10-02 | 複数のctp付加を有するホルモンアナログ |
JP2007306928A Pending JP2008073057A (ja) | 1991-10-04 | 2007-11-28 | 複数のctp付加を有するホルモンアナログ |
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EP (1) | EP0607297B1 (ja) |
JP (2) | JP4081137B2 (ja) |
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1992
- 1992-10-02 WO PCT/US1992/008424 patent/WO1993006844A1/en active IP Right Grant
- 1992-10-02 EP EP92921759A patent/EP0607297B1/en not_active Expired - Lifetime
- 1992-10-02 JP JP50709393A patent/JP4081137B2/ja not_active Expired - Lifetime
- 1992-10-02 CA CA002120358A patent/CA2120358A1/en active Granted
- 1992-10-02 DE DE69233188T patent/DE69233188T2/de not_active Expired - Lifetime
- 1992-10-02 AT AT92921759T patent/ATE248922T1/de not_active IP Right Cessation
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- 1992-10-02 KR KR1019940701089A patent/KR100281811B1/ko not_active IP Right Cessation
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JP4081137B2 (ja) | 2008-04-23 |
EP0607297B1 (en) | 2003-09-03 |
JPH07502166A (ja) | 1995-03-09 |
ATE248922T1 (de) | 2003-09-15 |
WO1993006844A1 (en) | 1993-04-15 |
DE69233188T2 (de) | 2004-05-27 |
NO941186L (no) | 1994-03-30 |
AU667489B2 (en) | 1996-03-28 |
ES2204893T3 (es) | 2004-05-01 |
FI941500A0 (fi) | 1994-03-30 |
NO941186D0 (no) | 1994-03-30 |
AU2864892A (en) | 1993-05-03 |
US6225449B1 (en) | 2001-05-01 |
CA2120358A1 (en) | 1993-04-15 |
EP0607297A4 (en) | 1995-10-25 |
FI941500A (fi) | 1994-05-23 |
DE69233188D1 (de) | 2003-10-09 |
KR100281811B1 (ko) | 2001-02-15 |
EP0607297A1 (en) | 1994-07-27 |
US5759818A (en) | 1998-06-02 |
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