JP2021505135A - Cdkl5発現変異体及びcdkl5融合タンパク質 - Google Patents
Cdkl5発現変異体及びcdkl5融合タンパク質 Download PDFInfo
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Abstract
Description
本明細書で使用される「CDKL5媒介性神経障害」は、CDKL5タンパク質の発現又は過剰発現によって処置され得る任意の疾患又は障害を指す。
ヒトCDKL5遺伝子は、24個のエクソンから構成され、そのうち最初の3個は(エクソン1、1a、及び1b)は翻訳されていない。
図1Aは、CDKL5107のポリペプチドマップを示す。野生型全長ヒトCDKL5107アイソフォームのアミノ酸配列は、配列番号1で示されている。CDKL5107タンパク質は、960個のアミノ酸からなり、キナーゼドメインは、最初の約300個のアミノ酸に含まれている。960のうちの残基42は、リン酸化反応中にATP結合に関与するキナーゼドメイン内にある重要なリジン残基であり、この残基の変異は、一般にキナーゼ活性の喪失(「キナーゼデッド(Kinase dead)」)をもたらす。さらに、2つの核局在化シグナルが、残基312〜315(NLS1)と784〜789(NLS2)にまたがって存在し、核外輸送シグナル(NES)が、残基836〜845にまたがって存在する。残基905〜960にまたがったC末端のアミノ酸は、CDKL5107に固有であり、CDKL5115には存在しない。アミノ酸残基1〜904は、CDKL5115とCDKL5107との間で同一である。野生型完全長ヒトCDKL5115アイソフォームのアミノ酸配列は、配列番号47で示されている。
様々なウイルス及び細胞タンパク質は、細胞膜を通過する移動を媒介する塩基性ポリペプチド配列を有する。細胞膜を通過して移動する能力は、膜を通過して高分子量ポリペプチドを送達するための重要なツールとなっている。「タンパク質形質導入ドメイン」(PTD)及び「細胞透過性ペプチド」(CPP)という語句は、通常は、すべてではないにしても、多くの哺乳動物細胞の原形質膜を通過できる短いペプチド(30アミノ酸未満)を指すために使用される。それらが原形質膜を集団で通過するのを可能にするドメインの特定の特性を特定する研究の後、研究者らは、これらのドメインがリジン及びアルギニンなどの塩基性アミノ酸残基を多数含むことを観察した。従って、細胞透過性ペプチドは、2つのクラスに分類され:第1のクラスは、正の電荷に寄与するリジン残基を含む両親媒性ヘリックスペプチドからなるが、第2のクラスは、アルギニンリッチペプチドを含む。これらのペプチドは、細胞内標的に送達するのが難しい他のタンパク質と組み合わせて使用される場合、治療の可能性を有し得る。PTDの最も頻繁な実験的使用は、TAT、アンテナペディア(Antp)、及び他のポリ−アルギニンペプチドである。
上記のように、CDKL5変異体は、CPPも含むタンパク質などの融合タンパク質に使用することができる。タンパク質分泌を増強するためのリーダーシグナルポリペプチド又は融合タンパク質を検出及び/又は精製するためのタグ、並びに機能的ポリペプチドを連結するために使用できるリンカーポリペプチドなどの他のポリペプチドもまた、そのような融合タンパク質に組み込むことができる。
組換えタンパク質(例えば、CDKL5変異体又は融合タンパク質)は、ヒトへの投与に適合された医薬組成物として通常の手順に従って製剤化することができる。例えば、1つ又は複数の実施形態では、静脈内投与用の組成物は、無菌等張水性緩衝液中の溶液である。必要に応じて、この組成物は、可溶化剤及び注射部位の痛みを和らげるための局所麻酔剤も含み得る。一般に、成分は、個別に、又は単位剤形中に一緒に混合されて、例えば、活性物質の量を表示するアンプル又は小袋などの密閉容器内の乾燥凍結乾燥粉末又は無水濃縮物として供給される。組成物が注入によって投与される場合、医薬品グレードの滅菌水、生理食塩水、又はデキストロース/水を含む点滴ボトルで注入することができる。組成物が注射によって投与される場合、投与前に成分を混合できるように、注射用滅菌水又は生理食塩水のアンプルを提供することができる。
組換えタンパク質(例えば、CDKL5変異体又は融合タンパク質)は、適切なベクターを使用して宿主細胞で発現させ、分泌させることができる。例えば、哺乳動物細胞(例えば、CHO、HeLa、若しくはHEK細胞)又は細菌細胞(例えば、大腸菌(Escherichia coli若しくはP.ハロプランクティス(P.haloplanktis)TAC 125細胞)を使用することができる。例示的なプラスミドは、以下の実施例に記載されており、図2A〜図2ADに示されている。当業者であれば、細胞の形質転換、トランスフェクション、又は形質導入に適した代替ベクターを選択して、本明細書に記載のCDKL5変異体及び融合タンパク質を産生させることができる。
図2A〜図2ADは、哺乳動物細胞(例えば、CHO細胞)又は細菌細胞(例えば、E.coli細胞)などの適切な細胞において融合タンパク質を発現させるためのプラスミドを示す。これらのタンパク質は、配列番号19〜46に示されるアミノ酸配列を有する。欠失又は切断の付番は、完全長CDKL5107ポリペプチド(1〜960)に関連している。CDKL5が追加のN末端アミノ酸配列にC末端で融合している構成物では、CDKL5の最初のメチオニン(アミノ酸1)が除去されている。これらの構成物では、CDKL5ポリペプチドは、2番目のアミノ酸であるリジンで始まる。図2A〜図2AD並びに配列番号19〜46及び54〜55で使用される略語は、以下の表1に要約されている:
Claims (44)
- 配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9、配列番号10、配列番号11、又は配列番号12に対して少なくとも98%の配列同一性を有する配列を含むCDKL5ポリペプチド。
- 配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9、配列番号10、配列番号11、又は配列番号12に対して少なくとも99%の配列同一性を有する配列を含む、請求項1に記載のCDKL5ポリペプチド。
- 配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9、配列番号10、配列番号11、又は配列番号12に対して100%の配列同一性を有する配列を含む、請求項1に記載のCDKL5ポリペプチド。
- 核外輸送シグナル(NES)を欠くCDKL5ポリペプチド。
- 核局在化シグナル(NLS)を含む、請求項4に記載のCDKL5ポリペプチド。
- 核局在化シグナル(NLS)を含まない、請求項4に記載のCDKL5ポリペプチド。
- 核局在化シグナル(NLS)を欠き、核外輸送シグナル(NES)を含むCDKL5ポリペプチド。
- 請求項1〜7のいずれか一項に記載のCDKL5ポリペプチド、及び前記CDKL5ポリペプチドに機能的に結合されたリーダーシグナルポリペプチドを含む融合タンパク質。
- 請求項1〜7のいずれか一項に記載のCDKL5ポリペプチド、及び前記CDKL5ポリペプチドに機能的に結合された細胞透過性ポリペプチドを含む融合タンパク質。
- 前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、又は配列番号50に対して少なくとも90%の配列同一性を有する配列を含む、請求項9に記載の融合タンパク質。
- 前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、又は配列番号50に対して100%の配列同一性を有する配列を含む、請求項9に記載の融合タンパク質。
- 請求項9〜11のいずれか一項に記載の融合タンパク質に機能的に結合されているリーダーシグナルポリペプチドをさらに含む、請求項9〜11のいずれか一項に記載の融合タンパク質。
- 前記リーダーシグナルポリペプチドが、配列番号48、配列番号49、配列番号51、配列番号52、又は配列番号53に対して少なくとも90%の配列同一性を有する配列を含む、請求項8又は12に記載の融合タンパク質。
- 前記リーダーシグナルポリペプチドが、配列番号48、配列番号49、配列番号51、配列番号52、又は配列番号53に対して100%の配列同一性を有する配列を含む、請求項8又は12に記載の融合タンパク質。
- 請求項1〜7のいずれか一項に記載のCDKL5ポリペプチド又は請求項8〜14のいずれか一項に記載の融合タンパク質;及び
薬学的に許容される担体を含む、医薬製剤。 - 請求項15に記載の製剤を、それを必要とする患者に投与するステップを含む、CDKL5媒介性神経障害を処置する方法。
- 前記製剤が、くも膜下腔内、静脈内、大槽内、脳室内、又は実質内に投与される、請求項16に記載の方法。
- 前記製剤が、くも膜下腔内又は静脈内に投与される、請求項16又は17に記載の方法。
- 前記CDKL5媒介性神経障害が、CDKL5欠損症又はCDKL5変異若しくは欠損によって引き起こされる非定型レット症候群の1つ又は複数である、請求項16〜18のいずれか一項に記載の方法。
- 請求項1〜7のいずれか一項に記載のCDKL5ポリペプチド又は請求項8〜14のいずれか一項に記載の融合タンパク質を製造する方法であって:
前記CDKL5ポリペプチド又は前記融合タンパク質を発現させるステップ;及び
前記CDKL5ポリペプチド又は前記融合タンパク質を精製するステップを含む、方法。 - 前記CDKL5ポリペプチド又は前記融合タンパク質が、チャイニーズハムスター卵巣(CHO)細胞、HeLa細胞、ヒト胚腎臓(HEK)細胞、又は大腸菌(Escherichia coli)細胞で発現される、請求項20に記載の方法。
- 請求項1〜7のいずれか一項のCDKL5ポリペプチド又は請求項8〜14のいずれか一項の融合タンパク質をコードするポリヌクレオチド。
- 請求項22に記載のポリヌクレオチドを含むベクター。
- 共に機能的に結合されたCDKL5ポリペプチド及び細胞透過性ポリペプチドを含む融合タンパク質であって、前記CDKL5ポリペプチドが、配列番号1又は配列番号47に対して少なくとも98%の配列同一性を有する配列を含み、前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号17、配列番号18、又は配列番号50に対して少なくとも90%の配列同一性を有する配列を含む、融合タンパク質。
- 前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号17、配列番号18、又は配列番号50に対して少なくとも95%の配列同一性を有する配列を含む、請求項24に記載の融合タンパク質。
- 前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号17、配列番号18、又は配列番号50に対して100%の配列同一性を有する配列を含む、請求項25に記載の融合タンパク質。
- リーダーシグナルポリペプチドをさらに含む、請求項24〜26のいずれか一項に記載の融合タンパク質。
- 前記リーダーシグナルポリペプチドが、配列番号48、配列番号49、配列番号51、配列番号52、又は配列番号53に対して少なくとも90%の配列同一性を有する配列を含む、請求項27に記載の融合タンパク質。
- 前記リーダーシグナルポリペプチドが、配列番号48、配列番号49、配列番号51、配列番号52、又は配列番号53に対して100%の配列同一性を有する配列を含む、請求項28に記載の融合タンパク質。
- 共に機能的に結合されたCDKL5ポリペプチド及びリーダーシグナルポリペプチドを含む融合タンパク質であって、前記CDKL5ポリペプチドが、配列番号1又は配列番号47に対して少なくとも98%の配列同一性を有する配列を含み、前記リーダーシグナルポリペプチドが、配列番号48、配列番号51、配列番号52、又は配列番号53に対して少なくとも90%の配列同一性を有する配列を含む、融合タンパク質。
- 前記リーダーシグナルポリペプチドが、配列番号48、配列番号51、配列番号52、又は配列番号53に対して100%の配列同一性を有する配列を含む、請求項30に記載の融合タンパク質。
- 細胞透過性ポリペプチドをさらに含む、請求項30又は31に記載の融合タンパク質。
- 前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、又は配列番号50に対して少なくとも90%の配列同一性を有する配列を含む、請求項32に記載の融合タンパク質。
- 前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、又は配列番号50に対して少なくとも95%の配列同一性を有する配列を含む、請求項33に記載の融合タンパク質。
- 前記細胞透過性ポリペプチドが、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、又は配列番号50に対して100%の配列同一性を有する配列を含む、請求項34に記載の融合タンパク質。
- 請求項24〜35のいずれか一項に記載の融合タンパク質;及び薬学的に許容される担体を含む医薬製剤。
- CDKL5媒介性神経障害を処置する方法であって、請求項36に記載の製剤を、それを必要とする患者に投与するステップを含む、方法。
- 前記製剤が、くも膜下腔内、静脈内、大槽内、脳室内、又は実質内に投与される、請求項37に記載の方法。
- 前記製剤が、くも膜下腔内又は静脈内に投与される、請求項37又は38に記載の方法。
- 前記CDKL5媒介性神経障害が、CDKL5欠損症又はCDKL5変異若しくは欠損によって引き起こされる非定型レット症候群の1つ又は複数である、請求項37〜39のいずれか一項に記載の方法。
- 請求項24〜35のいずれか一項に記載の融合タンパク質を製造する方法であって:
前記融合タンパク質を発現させるステップ;及び
前記融合タンパク質を精製するステップを含む、方法。 - 前記融合タンパク質が、チャイニーズハムスター卵巣(CHO)細胞、HeLa細胞、ヒト胚腎臓(HEK)細胞、又は大腸菌(Escherichia coli)細胞で発現される、請求項41に記載の方法。
- 請求項24〜35のいずれか一項に記載の融合タンパク質をコードするポリヌクレオチド。
- 請求項43に記載のポリヌクレオチドを含むベクター。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008151388A1 (en) * | 2007-06-15 | 2008-12-18 | Cytomatrix Pty Ltd | Methods of initiating insulin production |
WO2014001572A1 (en) * | 2012-06-29 | 2014-01-03 | Lysando Ag | Composition for use in mycobacteria therapy |
JP2014503198A (ja) * | 2010-11-22 | 2014-02-13 | カリダス・バイオファーマ,インコーポレーテッド | 組換え酵素および他のタンパク質のタンパク質発現および分泌を改善する新規シグナル配列 |
CN104086656A (zh) * | 2014-03-27 | 2014-10-08 | 华东理工大学 | 一种TAT-RhoGDI2融合蛋白及其制备方法和应用 |
WO2015031673A2 (en) * | 2013-08-28 | 2015-03-05 | Bioasis Technologies Inc. | Cns-targeted conjugates having modified fc regions and methods of use thereof |
JP2017507655A (ja) * | 2014-02-28 | 2017-03-23 | アルマ マータ ステューディオラム — ユニバーシタ ディ ボローニャ | TATk−CDKL5融合タンパク質、組成物、製剤、及びそれらの使用 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008151388A1 (en) * | 2007-06-15 | 2008-12-18 | Cytomatrix Pty Ltd | Methods of initiating insulin production |
JP2014503198A (ja) * | 2010-11-22 | 2014-02-13 | カリダス・バイオファーマ,インコーポレーテッド | 組換え酵素および他のタンパク質のタンパク質発現および分泌を改善する新規シグナル配列 |
WO2014001572A1 (en) * | 2012-06-29 | 2014-01-03 | Lysando Ag | Composition for use in mycobacteria therapy |
WO2015031673A2 (en) * | 2013-08-28 | 2015-03-05 | Bioasis Technologies Inc. | Cns-targeted conjugates having modified fc regions and methods of use thereof |
JP2017507655A (ja) * | 2014-02-28 | 2017-03-23 | アルマ マータ ステューディオラム — ユニバーシタ ディ ボローニャ | TATk−CDKL5融合タンパク質、組成物、製剤、及びそれらの使用 |
CN104086656A (zh) * | 2014-03-27 | 2014-10-08 | 华东理工大学 | 一种TAT-RhoGDI2融合蛋白及其制备方法和应用 |
Non-Patent Citations (8)
Title |
---|
BIOCHEM. BIOPHYS. RES. COMMUN., vol. 482, JPN6022045060, 8 January 2017 (2017-01-08), pages 239 - 245, ISSN: 0004904547 * |
BIOCHIM. BIOPHYS. ACTA, vol. 1805, JPN6022045058, 2010, pages 25 - 34, ISSN: 0004904545 * |
J. BIOL. CHEM., vol. 281, no. 42, JPN6022045059, 2006, pages 32048 - 32056, ISSN: 0004904546 * |
J. BIOL. CHEM., vol. 281, no. 48, JPN6022045055, 2006, pages 36579 - 36587, ISSN: 0004904543 * |
J. BIOL. CHEM., vol. 284, no. 11, JPN6022045057, 2009, pages 7376 - 7384, ISSN: 0004904544 * |
NEURAL PLAST., vol. Vol. 2012, Article ID 728267, JPN6022045053, 2012, pages 1 - 11, ISSN: 0004904541 * |
PLOS ONE, vol. Vol. 11: e0157758, JPN6022045062, 2016, pages 1 - 22, ISSN: 0004904548 * |
PLOS ONE, vol. Vol. 7, No. 2, e30846, JPN6022045054, 2012, pages 1 - 14, ISSN: 0004904542 * |
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