JP4734251B2 - チロシナーゼ突然変異体及びその使用方法 - Google Patents
チロシナーゼ突然変異体及びその使用方法 Download PDFInfo
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- JP4734251B2 JP4734251B2 JP2006534327A JP2006534327A JP4734251B2 JP 4734251 B2 JP4734251 B2 JP 4734251B2 JP 2006534327 A JP2006534327 A JP 2006534327A JP 2006534327 A JP2006534327 A JP 2006534327A JP 4734251 B2 JP4734251 B2 JP 4734251B2
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- tyrosinase
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Description
ヒトチロシナーゼはI型膜糖タンパク質であり、529個のアミノ酸、7個の結合N−グリコシル化部位、2つのシステインに富むドメインに分類される17個のシステイン残基、2つの銅結合ドメイン及び1つのC末端TMドメインがある(Ujvari et al., J. Biol. Chem, 276(8):5924-3, 2001)。本発明者らは、膜貫通(TM)ドメインがないトランケート型ヒトチロシナーゼを構築した。TMドメインがない場合、ER内腔の鎖は天然高次構造へと折りたたまれなかった。しかし、翻訳速度を低下させると、活性タンパク質を生じる、トランケート型鎖の生産的折りたたみが起こることが示された。酵素的に活性な可溶性チロシナーゼは低温でも生産され、どちらの場合も、生産的折りたたみは初期段階のCNX相互作用に関連していた。この証拠により、フォールディング及び鎖をトランスロコン環境に保持し、それによりそのCNXとの相互作用を促進することにおけるTMドメインの役割が裏付けられた。
本発明では、CTL免疫応答を惹起するのに適した可溶性チロシナーゼ突然変異体をコードするポリヌクレオチド及び場合により製薬上適切な賦形剤を含む、免疫原性組成物を考慮する。標的細胞への免疫原性組成物の送達後、発現された本発明のチロシナーゼは小胞体内に保持され、分解されて、その後抗原提示のためにMHCクラスIにより細胞表面に提示される。好ましくは、可溶性チロシナーゼ突然変異体は膜貫通ドメインを欠く。最も好ましくは、前記可溶性チロシナーゼ突然変異体は配列番号1に示す核酸配列又はその変異体を含む。
本発明はまた、修飾チロシナーゼcDNAを投与することを含む、黒色腫を治療するための方法を開示する。ここに記載する修飾チロシナーゼは、トランスフェクト/形質導入抗原提示細胞(APC)のER内に残留する可溶性チロシナーゼ突然変異体である。このタンパク質はその後プロセシングされ、それに由来する抗原性ペプチドはAPC上のHLA分子と複合体を形成する。これらの複合体は、その後、溶解のために異常細胞を標的する細胞傷害性T細胞に認識される。
切断型ヒトチロシナーゼを構築することを含む、可溶性チロシナーゼ突然変異体を作製するための方法をここに記載する。好ましい実施形態では、チロシナーゼ突然変異体はカルネキシンに対する親和性が低い。また、チロシナーゼ突然変異体は膜貫通ドメインを欠失及び/又は少なくとも1つのグリコシル化部位を欠失していることが好ましい。さらに好ましくは、チロシナーゼ突然変異体は、配列番号1のポリヌクレオチドでコードされるか又はその変異体である。
(実施例)
正プライマー:5’−GCTATACCATGGCCCTCCTGGCTGTTTTG−3’
WT逆プライマー:5’−GGCGCGCCTCGAGTAAATGGCTCTGATA−3’
ST逆プライマー:5’−GTATTCTCGAGCCGACTCGCTTGTTC−3’
を使用してPCRにより増幅した。
対数増殖期のCHO細胞をトランスフェクションのために6穴プレートで培養し、Lipofectamine Plus(Invitrogen)を用いてチロシナーゼcDNAを一過性発現するために用いた。細胞をトランスフェクションの24時間後に収集し、削り取ってペレット化した。代謝標識のために、トランスフェクトしたCHO細胞(107細胞/ml)をシステイン/メチオニン不含培地で1時間飢餓させ、100−150μCi[35S]システイン/メチオニンで20分間パルス標識して、規定された時間チェイスした。チェイス後すぐに、細胞を低温PBS中に取り、20mM N−エチルマレイミド(NEM)中で30分間インキュベートして、遊離スルフヒドリル基をアルキル化した。次に細胞をCHAPS溶解緩衝液(2%CHAPS、200mM NaCl及びロイペプチン、アプロチニン、EDTAナトリウム、ベスタチン、AEBSF及びE−64を含む0.5%プロテアーゼ阻害因子カクテル(Sigma)を含む50mM HEPES緩衝液、pH7.5)で溶解した。
[35S]標識細胞溶解産物を遠心分離し、上清をT311抗体(1:50)又は抗カルネキシン抗体(1:100)と共に4℃で一晩インキュベートした。次にプロテインAセファロース20μlを添加し、前記細胞溶解産物を4℃で1時間インキュベートした。スラリーをHEPES緩衝液中0.5%CHAPSで3回洗った。5%2−メルカプトエタノールを含む(還元条件)又は含まない(非還元条件)SDS試料緩衝液中でスラリーを5分間煮沸することによりチロシナーゼを溶出した。共免疫沈降試験のために、溶解産物を抗カルネキシン抗体(1:100)で免疫沈降させ、洗浄したスラリーを1%SDSで溶出して、溶解緩衝液で10倍希釈し、T311で再沈降させた。結合タンパク質を天然又は還元条件で溶出し、10%SDS−PAGEゲルで分離した。その後オートラジオグラフィーによりゲルを視覚化した。
DOPAオキシダーゼアッセイは、チロシナーゼの二次触媒活性、すなわちDOPAキニンによるL−DOPAからDOPAクロムへの変換を測定する。このアッセイを、L−DOPAを基質として使用してゲル中で実施した(Negroiu et al., 2000)。トランスフェクションの24時間後に収集したトランスフェクト細胞の粗溶解産物又は細胞培養培地を、天然条件下でSDS−PAGEにより泳動させ、2.5mg/ml L−DOPA中でインキュベートしてチロシナーゼ活性を視覚化した。
種々のcDNAでトランスフェクトした溶解CHO細胞からのタンパク質を、記述されているように(Branza-Nichita et al., 1999)10%アクリルアミドゲル中で電気泳動しって分離し、イモビロン膜(Amersham International,Amersham,UK)に転写した。
可溶性チロシナーゼ突然変異体の成熟をパルス−チェイス分析によりインビボで観測し、モノクローナル抗チロシナーゼ抗体(T311)で免疫沈降させた。試料を2つに分け、各々の試料の半分をEndoH制限酵素で消化して、還元SDS−PAGEゲル中で消化していない対照に続いて泳動させた(図1)。EndoHは高マンノース型及び混成型N−グリカンだけを消化するので、EndoHの感受性を使用して高マンノースから複合型構造へのグリカンの成熟を観測した。EndoHによる消化は、プールを55kDで泳動するポリペプチドに還元した。5時間のチェイス中、前駆体は同じ電気泳動移動度を有し、完全にEndoH感受性のままであって、そのN−グリカンがゴルジ体で複合型構造にプロセシングされなかったことを示した(図1、レーン1、3、5、7)。1時間の合成後、免疫沈降タンパク質の量の漸減傾向が認められた(図1)。
折りたたみが膜貫通ドメインの存在でどのような影響を受けるかを検討するため、I型膜糖タンパク質−チロシナーゼをモデルとして用いて、その折りたたみ経路を、TMドメインがない構築物の折りたたみ経路と比較した。チロシナーゼは、哺乳動物の色素合成を調節するメラニン産生酵素である(Petrescu et al., 1997)。本発明者らは以前、その折りたたみがグリコシル化に依存することを立証した(Branza-Nichita et al, 1999,2000)。
コンセンサス配列Asn−Arg−Thrがないチロシナーゼ突然変異体を86位に構築した。これは、Asn86をGlnに突然変異させ、それによりシークオンをGln−Arg−Thrに変えることにより実施した。突然変異型Tyrmut1のER残留をそのEndoH消化パターンにより図6に示す。
C型肝炎ウイルスエンベロープタンパク質(HCV E2)膜貫通ドメインとチロシナーゼエクトドメインを用いてチロシナーゼキメラタンパク質(TyrE2)を構築した。図7に示すように、TyrE2キメラを発現する細胞溶解産物のEndoH消化により、ER残留プロフィールを有するタンパク質が生じた。
付加的な実施形態は本発明の範囲内である。例えば、以下の番号を付した実施形態により本発明をさらに例示する:
1.可溶性チロシナーゼ突然変異体を含み、前記チロシナーゼ突然変異体が小胞体内に蓄積しうる、ポリペプチド。
2.前記可溶性チロシナーゼ突然変異体がカルネキシンに対する親和性が低い、実施形態1に記載のチロシナーゼ突然変異体。
3.前記可溶性チロシナーゼ突然変異体が膜貫通ドメインを欠く、実施形態2に記載のチロシナーゼ突然変異体。
4.前記可溶性チロシナーゼ突然変異体が、配列番号1のポリヌクレオチドによりコードされるか又はその変異体である、実施形態2に記載のチロシナーゼ突然変異体。
5.前記可溶性チロシナーゼ突然変異体が少なくとも1つのグリコシル化部位を欠く、実施形態2に記載のチロシナーゼ突然変異体。
6.小胞体内に蓄積しうる可溶性チロシナーゼ突然変異体を含む免疫原性組成物。
7.前記可溶性チロシナーゼ突然変異体が、配列番号1のポリヌクレオチドによりコードされるか又はその変異体である、実施形態6に記載の免疫原性組成物。
8.小胞体内に蓄積しうる可溶性チロシナーゼ突然変異体である黒色腫抗原をコードするポリヌクレオチド。
9.前記可溶性チロシナーゼ突然変異体が膜貫通ドメインを欠く、実施形態8に記載のポリヌクレオチド。
10.前記可溶性チロシナーゼ突然変異体が、配列番号1において特定される配列又はその変異体によりコードされる、実施形態9に記載のポリヌクレオチド。
11.可溶性チロシナーゼ突然変異体をコードするポリヌクレオチド及び製薬上許容される担体を含むワクチン。
12.前記ポリヌクレオチドが、配列番号1において特定される配列又はその変異体を含む、実施形態11に記載のワクチン。
13.可溶性チロシナーゼ突然変異体をコードするポリヌクレオチドを含む宿主細胞。
14.前記ポリヌクレオチドが、配列番号1に示す配列又はその変異体を含む、実施形態13に記載の宿主細胞。
15.可溶性チロシナーゼ突然変異体をコードするポリヌクレオチドを抗原提示細胞に投与すること及び細胞傷害性リンパ球免疫応答を惹起することを含む、黒色腫を治療するための方法。
16.前記可溶性チロシナーゼ突然変異体が細胞の小胞体内に蓄積する、実施形態15に記載の方法。
17.前記可溶性チロシナーゼ突然変異体が膜貫通ドメインを欠く、実施形態16に記載の方法。
18.トランケート型のヒトチロシナーゼを構築することを含む、膜貫通ドメインを欠く可溶性チロシナーゼ突然変異体を作製するための方法。
本明細書において引用する全ての出版物及び特許出願及び特許は、それらの全体が参照してここに組み込まれる。
Claims (17)
- 小胞体内に蓄積でき、かつ(a)膜貫通ドメインの欠失および(b)86位におけるAsnへの突然変異導入によるコンセンサス配列Asn−Arg−Thrの欠失のうち少なくとも1つを含むチロシナーゼ突然変異体を含むポリペプチド。
- 前記チロシナーゼ突然変異体がカルネキシンに対する親和性が低い、請求項1に記載のポリペプチド。
- 前記チロシナーゼ突然変異体が、配列番号1のポリヌクレオチドによりコードされるか又はその変異体である、請求項2に記載のポリペプチド。
- 小胞体内に蓄積でき、かつ(a)膜貫通ドメインの欠失および(b)86位におけるAsnへの突然変異導入によるコンセンサス配列Asn−Arg−Thrの欠失のうち少なくとも1つを含むチロシナーゼ突然変異体を含む免疫原性組成物。
- 前記チロシナーゼ突然変異体が、配列番号1のポリヌクレオチドによりコードされるか又はその変異体である、請求項4に記載の免疫原性組成物。
- 小胞体内に蓄積でき、かつ(a)膜貫通ドメインの欠失、および(b)86位におけるAsnへの突然変異導入によるコンセンサス配列Asn−Arg−Thrの欠失のうち少なくとも1つを含むチロシナーゼ突然変異体をコードするポリヌクレオチド。
- 前記チロシナーゼ突然変異体が、配列番号1において特定される配列又はその変異体によりコードされる、請求項6に記載のポリヌクレオチド。
- チロシナーゼ突然変異体をコードする請求項6に記載のポリヌクレオチド及び製薬上許容される担体を含むワクチン。
- 前記ポリヌクレオチドが、配列番号1において特定される配列又はその変異体を含む、請求項8に記載のワクチン。
- チロシナーゼ突然変異体をコードする請求項6に記載のポリヌクレオチドを含む宿主細胞。
- 前記ポリヌクレオチドが、配列番号1に示す配列又はその変異体を含む、請求項10に記載の宿主細胞。
- チロシナーゼ突然変異体をコードする請求項6に記載のポリヌクレオチドを含む、抗原提示細胞に投与すること及び細胞傷害性リンパ球免疫応答を惹起することによって黒色腫を治療するための、組成物。
- 前記ポリヌクレオチドが配列番号1に示す配列またはその変異体を含む、請求項12に記載の組成物。
- トランケート型のヒトチロシナーゼを構築することを含み、小胞体内に蓄積でき、かつ(a)膜貫通ドメインの欠失および(b)86位におけるAsnへの突然変異導入によるコンセンサス配列Asn−Arg−Thrの欠失のうち少なくとも1つを含む、チロシナーゼ突然変異体を作製するための方法。
- 前記チロシナーゼ突然変異体がチロシナーゼキメラである、請求項1に記載のポリペプチド。
- 前記チロシナーゼキメラがもう1つ別のタンパク質の膜貫通ドメインを通して膜結合しており、前記膜貫通ドメインがER残留シグナルを含む、請求項15に記載のポリペプチド。
- 前記チロシナーゼキメラが、C型肝炎エンベロープタンパク質2の膜貫通ドメインにおける残留シグナルを通してER内に残留する、請求項16に記載のポリペプチド。
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WO2018024110A1 (zh) | 2016-08-01 | 2018-02-08 | 北京大学 | Mg53突变体及其制备方法和用途 |
KR102022611B1 (ko) * | 2016-12-29 | 2019-09-18 | 충남대학교 산학협력단 | 신규 재조합 티로시나아제 |
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US5605793A (en) * | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
US6472375B1 (en) * | 1998-04-16 | 2002-10-29 | John Wayne Cancer Institute | DNA vaccine and methods for its use |
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US20050142143A1 (en) | 2005-06-30 |
EP1689430A2 (en) | 2006-08-16 |
CN1863552A (zh) | 2006-11-15 |
ATE437650T1 (de) | 2009-08-15 |
CN1863552B (zh) | 2012-06-27 |
WO2005035723A2 (en) | 2005-04-21 |
CA2541833A1 (en) | 2005-04-21 |
DE602004022323D1 (de) | 2009-09-10 |
ES2329904T3 (es) | 2009-12-02 |
JP2007507240A (ja) | 2007-03-29 |
US20120034675A1 (en) | 2012-02-09 |
WO2005035723A3 (en) | 2006-01-19 |
EP1689430B1 (en) | 2009-07-29 |
EP1689430A4 (en) | 2007-08-29 |
KR20060114326A (ko) | 2006-11-06 |
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