JP6787651B2 - B−Rafキナーゼ阻害剤に対する感受性についての診断試験 - Google Patents
B−Rafキナーゼ阻害剤に対する感受性についての診断試験 Download PDFInfo
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- JP6787651B2 JP6787651B2 JP2015094227A JP2015094227A JP6787651B2 JP 6787651 B2 JP6787651 B2 JP 6787651B2 JP 2015094227 A JP2015094227 A JP 2015094227A JP 2015094227 A JP2015094227 A JP 2015094227A JP 6787651 B2 JP6787651 B2 JP 6787651B2
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Description
本発明は、B-Rafキナーゼ阻害剤を用いた治療の候補者である患者の検出のための方法及び組成物を提供する。従って、一つの観点において、本発明は、B-Raf阻害剤に対する癌細胞の感受性を決定する方法であって:(i) 癌患者由来の癌細胞から核酸試料を準備し、(ii) 前記核酸試料中の標的ポリヌクレオチド配列を、当該標的ポリヌクレオチド配列を増幅するプライマー対を用いて増幅し、ここで、当該標的ポリヌクレオチド配列は、BRAFにV600E突然変異部位を含んで成り、そして増幅は、配列番号1に記載の配列のうちの少なくとも15個連続のヌクレオチドを含んで成り、且つBRAFのV600E突然変異部位にある突然変異配列の存在を検出する標識オリゴヌクレオチドプローブの存在下で実施される;そして(iii) BRAFにおけるV600E突然変異の有無を検出し;それによりB-Raf阻害剤に対する癌の感受性を決定すること、を含んで成る方法、を提供する。態様によっては、前記プローブは配列番号1に記載のヌクレオチド配列を有する。前記増幅は、V600E突然変異部位での野生型配列の存在を検出する第二プローブの存在下実施することができる。態様によっては、前記第二プローブは、配列番号2の少なくとも15個のヌクレオチドを含んで成る。態様によっては、前記プローブは、蛍光標識で標識することができる。前記プローブは更に、一方が蛍光色素であり、他方がクエンチング部分であるという2つの標識を用いて標識することもできる。
定義
本願の文脈において、「V600E」とはB-Rafのアミノ酸位600にあるバリンの代わりにグルタミンの置換をもたらすBRAFにおける突然変異(ヌクレオチド位1799のT>A)を意味する。「V600E」はまた、従来の付番方式のもとでは「V599E」(1796T>A)としても知られている(Kumar et al., Clin. Cancer Res. 9:3362-3368, 2003).。
本発明は、癌患者、例えば黒色腫の癌患者、結腸癌患者、甲状腺癌患者、及び低悪性度の重度の卵巣癌を有する患者であって、B-Raf阻害剤、例えば選択的突然変異B-Raf阻害剤、例えばPLX4032による治療の候補者である患者を選択するのに使用するためのV600E診断アッセイを提供する。従って、本診断試験は、PLX4032による治療に応答する可能性に従うことで患者を分類するのに使用することができる。
V600E突然変異は、様々な種類の癌、例えば黒色腫、結腸直腸癌;甲状腺癌、例えば甲状腺乳頭癌;及び卵巣癌、例えば低悪性度の重度の癌で検出することができる。態様によっては、V600E突然変異は、肺癌、神経膠腫、前立腺癌、肺癌、肉腫、肝臓癌、下垂体癌、並びに大腸、胆道、眼、膵臓、胃、中枢神経系、造血組織及びリンパ系組織で生じる癌において検出される。
V600E突然変異の存在について核酸を評価するための検出技術は、分子遺伝学の分野で周知の手順を伴う。更に、この方法の多くが核酸の増幅を伴う。かかる手順を実施するための十分な指針が当業界で提供されている。文献の例として、例えばPCR Technology:Principles and Applications for DNA Amplication (H. A. Erlich編, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Aplications (Innisら編, Academic Press, San Diego, Calif., 1990); Current Protocols in Molecular Biology, Ausubel, 1994-1999, 2004年4月の補遺版を含む; Sambrook及び& Russel, Molecular Cloning, A Laboratory Manual (第3版, 2001)がある。
対立遺伝子特異的ハイブリダイゼーションは、突然変異配列の一方に特異的なオリゴヌクレオチドを、核酸試料を増幅することで得られた増幅産物とハイブリダイズさせることにより、少なくとも1つの塩基が異なっている2つのDNA分子間での識別に依拠している。対立遺伝子特異的アッセイは、2つの対立遺伝子特異的オリゴヌクレオチド、例えば、一方の変異体と、他方の変異体に対し、別個にハイブリダイズする、最初の変異体用の対立遺伝子特異的プローブ及び第二の変異体用の対立遺伝子特異的プローブを含んで成ることがある。対立遺伝子特異的ハイブリダイゼーションは、典型的には、短いオリゴヌクレオチド、例えば15−35個の長さのヌクレオチドを利用する。かかるプローブを設計するための原理及びガイダンスは当業界で入手可能なものであり、例えば、本明細書で引用する文献に記載されている。ハイブリダイゼーション条件は、十分にストリンジェントであるべきであり、これは、対立遺伝子間でハイブリダイゼーション強度に有意な違いがあること、好ましくは本質的に二種類の応答に有意な違いがあって、プローブが当該対立遺伝子の一方にのみハイブリダイズする条件のことである。プローブによっては、標的DNAの1セグメントとハイブリダイズし、その結果、注目の部位、すなわちBRAFのエキソン15のコドン600におけるヌクレオチド位1799が、前記プローブの中心部(例えば、15塩基のオリゴヌクレオチドではその第7位;16塩基のオリゴヌクレオチドではその第8又は9位)とアラインするが、このデザインは必須ではない。
態様によっては、核酸試料は、米国特許第 5,210,015号明細書;同 5,487,972号明細書;及び同 5,804,375号明細書;及びHolland ら, 1988, Proc. Natl. Acad. Sci. USA 88: 7276-7280「TaqMan(登録商標)」又は「5’−ヌクレアーゼアッセイ」を用いて、V600E突然変異の存在についてアッセイされる。TaqMan(登録商標)アッセイでは、増幅された領域内でハイブリダイズする標識済みの検出プローブが増幅反応中存在している。プローブは、当該プローブがDNA合成のためのプライマーとして働かないように修飾される。増幅は、5’→3’エキソヌクレアーゼ活性を有するDNAポリメラーゼを用いて実施される。増幅の各合成ステップでは、伸張されるプライマーから下流の標的核酸にハイブリダイズする任意のプローブは、DNAポリメラーゼの5’→3’エキソヌクレアーゼ活性によって分解される。従って、新しい標的鎖の合成はプローブの分解をもたらし、この分解産物の蓄積は、標的配列の合成の指標となる。
TTS068-BRAF_F1: 5' CCTCACAGTAAAAATAGGTGATTTTGGTCTE 3' (E= t-ブチルベンジルdA) (配列番号25)
RL_BRAF_R5: 5' TAGCCTCAATTCTTACCATCCACAAAA 3' (配列番号4).
5' A AAT AGC CTC AAT TCT TAC CAT CCA CAA AA 3' (配列番号12)
5' TAG CCT CAA TTC TTA CCA TCC ACA AAA 3' (配列番号13)
5' TAG CCT CAA TTC TTA CCA TCC ACA AAE 3' (配列番号14)
5' AGG GCC AAA AAT TTA ATC AGT GGA AAA A 3' (配列番号15)
5' CAG TGG AAA AAT AGC CTC AAT TCT TAC CA 3' (配列番号16)
5' TTTCTTCATGAAGACCTCACAGTAAAAATE 3' (配列番号17);又は
5' ATATATTTCTTCATGAAGACCTCACAGTAAE 3' (配列番号18)
であってもよい。
5' ATG ACT TCT GGT GCC ATC CAC AA 3' (配列番号19)
を含んで成るプライマーを含んで成ることもできる。
5' AAA AAT AGC CTC AAT TCT TAC CAT CCA CAA AA 3' (配列番号20);
5' GCC ATC CAC AAA ATG GAT CCA GAC A 3' (配列番号21);又は
5' CAA AAT GGA TCC AGA CAA CTG TTC AAA 3' (配列番号22)
がある。
TTS155-BRAF_MU 5' QCTACAIAIFAAATCTCGATGGAGTGGGTCCCAP 3' (配列番号23)
TTS148-BRAF_WT 5' QACAITGEAAATCTCGATGGAGTGGGTCCCAP 3' (配列番号24)
(E = HEXレポーター色素, F= FAMレポーター色素, I =デオキシイノシン, Q = BHQ2クエンチャー色素, P= 3’-リン酸)。当該色素(F又はE)は、dIとdA(TTS155-BRAF_MU)又はdGとdA (TTS148-BRAF_WT)のいずれかの間に挿入される。
態様によっては、対立遺伝子特異的ハイブリダイゼーションは、固定された標的又は固定されたプローブを用いたアッセイフォーマットにおいて実施される。そのようなフォーマットは、当業界で知られており、例えば、ドット−ブロットフォーマット及びリバースドットブロットアッセイフォーマットがあり、これらは米国特許第5,310,893号;第5,451,512号;第5,468,613号;及び第5,604,099号に記載されている。
V600E突然変異の有無は、対立遺伝子特異的増幅又はプライマー伸長法を用いて検出することができる。これらの反応は、プライマーの3’末端、例えば3’ヌクレオチド又は3’ヌクレオチドの最後から2番目のヌクレオチドにあるミスマッチを介して突然変異(又は野生型)部位を特異的に標的にするよう設計されたプライマーの使用を通常伴う。ミスマッチの存在は、ポリメラーゼがエラー修正活性を欠いている場合、当該ポリメラーゼがプライマーを伸長する能力を発揮させる。例えば、対立遺伝子特異的増幅又は伸長をベースとした方法を用いてV600E突然変異配列を検出するために、BRAFのコドン600におけるヌクレオチド位1799にある突然変異A対立遺伝子と相補的なプライマーは、3’末端ヌクレオチドがこの突然変異の位置でハイブリダイズするよう設計されている。突然変異対立遺伝子の存在は、プライマーの伸長を開始する能力によって決定することができる。3’末端がミスマッチしている場合、伸長は阻害される。従って、例えば、プライマーが3’末端の突然変異対立遺伝子のヌクレオチドとミスマッチしている場合、プライマーは効果的に伸長する。増幅は、1799位にあるBRAF野生型配列由来の特異的な対立遺伝子特異的プライマーを用いて実施することもできる。
V600E突然変異の存在(不在)は、直接的な配列決定によっても検出することができる。方法として、ジデオキシ配列決定をベースとした方法及び、オリゴヌクレオチドの伸長産物のピロシーケンス (Pyrosequencing)(登録商標)のような方法がある。かかる方法は、しばしば、PCRのような増幅技術を利用する。配列決定のための他の類似の方法は、完全PCRの使用を必要としないが、典型的には、研究目的のヌクレオチドと相補的な1つの蛍光標識されたジデオキシリボ核酸分子(ddNTP)によるプライマーの伸長のみを典型的に使用する。多型部位にあるヌクレオチドは、一塩基伸長しているプライマーの検出を介して同定することができ、これは蛍光標識されている(例えば、Kobayashi et al, Mol. Cell. Probes, 9:175-182, 1995)。
B-Raf中のV600E突然変異は、野生型と突然変異のB-Rafを識別する方法によって検出することができる。しばしば、これらの方法は突然変異B-Rafと特異的な抗体を利用する。
突然変異の検出法には、ユーザーに対し、B-Raf V600E突然変異の有無に基づき、それぞれ患者がB-Raf阻害剤、例えば突然変異特異的B-Raf阻害剤、例えばPLX4032で治療されるべきか否かを知らせるデータ解析ソフトの使用も含まれる。
本発明は、本方法を実施するのに有用な成分を含んで成るキットを提供する。態様によっては、当該キットは、前記突然変異又はV600E対立遺伝子に特異的な1又は複数のオリゴヌクレオチドプローブを含んで成ることがある。かかるキットは、V600E突然変異部位を包含するBRAFの遺伝子座の領域を増幅する増幅プライマーを含むことができる。従って、態様によっては、キットは、プライマーセットとして
TTS068-BRAF_F1: 5' CCTCACAGTAAAAATAGGTGATTTTGGTCTE 3' (E= t-ブチルベンジルdA) (配列番号25)及び
RL_BRAF_R5: 5' TAGCCTCAATTCTTACCATCCACAAAA 3' (配列番号4)を含んで成り、これらは、BRAFの標的領域を増幅する。前記キットは、プローブ、例えば対立遺伝子特異的プローブを更に含んで成ることもできる。本発明のキットで使用することのできる対立遺伝子特異的プローブの例として:
TTS155-BRAF_MU 5' QCTACAIAIFAAATCTCGATGGAGTGGGTCCCAP 3' (配列番号23)
TTS148-BRAF_WT 5' QACAITGEAAATCTCGATGGAGTGGGTCCCAP 3' (配列番号24)
がある。
B-Raf V600E突然変異の検出
本実施例は、ホルマリン固定され、パラフィン包埋された(FFPE)癌の腫瘍組織から抽出したゲノムDNAのV600E(BRAFヌクレオチド1799T>A)突然変異の測定のためのリアルタイムPCR(ポリメラーゼ連鎖反応)ベースの診断試験を示すものである。本実施例において、FFPE組織から抽出したゲノムDNAは、TaqMan PCR解析によって試験した。PCR反応のために、Master MixをTaqMan RNA Reaction Mix, Primer-Probe Mix及び酢酸マグネシウム(Mg(OAc)2)補因子を混合することで調製した。増幅は、125 ngのゲノムDNAを用いて実施した。増幅産物は、COBAS TaqMan 48アナライザーを用いて検出した。
TTS068-BRAF_F1: 5' CCTCACAGTAAAAATAGGTGATTTTGGTCTE 3' (E= t-ブチルベンジルdA) (配列番号25)及び
RL_BRAF_R5: 5' TAGCCTCAATTCTTACCATCCACAAAA 3' (配列番号4)。
V600E (TTS155-BRAF_MU): 5' QCTACAIAIFAAATCTCGATGGAGTGGGTCCCAP 3' (配列番号23)
野生型(TTS148-BRAF_WT): 5' QACAITGEAAATCTCGATGGAGTGGGTCCCAP 3' (配列番号24)
(E = HEX レポーター色素, F= FAM レポーター色素, I =デオキシイノシン, Q = BHQ2クエンチャー色素, P= 3’-リン酸)。
本実験で利用するBRAFプライマー−プローブの混合物は、B-Raf WT又はV600Eをコードするヌクレオチド配列に特異的な二重標識された蛍光標識をそれぞれ含んでおり、これらは増幅プロセスの間、蛍光シグナルの放出を通じて特異的なPCR産物のリアルタイムな検出を可能にする。B-Raf WT及びV600Eプローブは、異なる蛍光レポーター色素及び同一のクエンチャー色素で標識されている。プローブが無傷の場合、レポーター色素の蛍光は、クエンチャー色素により、フォスター型のエネルギー変換に起因して抑制される。PCRの間、各プローブは、配列依存的に、その標的配列とハイブリダイズし、そしてZ05 DNAポリメラーゼの5’−ヌクレアーゼ活性によって開裂し、そして2つの色素を分離する。一旦レポーター及びクエンチャー色素が分離すると、レポーター色素由来の蛍光は検出可能になる。PCRサイクル毎にアンプリコンがより生成されるにつれ、レポーター色素由来の蓄積したシグナルは効果的に増大する。B-Raf WT及びV600E配列は、各サイクルの間各プローブをその相当の特異的な発光波長範囲で測定することで独立してモニタリングした。
B-Raf V600E(チャンネル1)及びWT(チャンネル2)のCt値は、COBAS TaqMan 48 Analyzerワークステーション上のAmpliLink 3.1ソフトウェアによって算出した。ネガティブ及びポジティブコントロール反応由来のCt値を用いて、ランが有効か否かを決定した。
表2は、各コントロール反応についての許容されるCt値を列挙している。
キーポイント: E = HEXレポーター色素、F= FAMレポーター色素、I =デオキシイノシン、Q = BHQ2クエンチャー色素、P= 3’-リン酸
配列番号1 BRAF_MU (I =デオキシイノシン P= 3’-リン酸)
5' CTACAIAIAAATCTCGATGGAGTGGGTCCCAP 3'
配列番号2 BRAF_WT (I =デオキシイノシン P= 3’-リン酸)
5' ACAITGAAATCTCGATGGAGTGGGTCCCAP 3'
配列番号3 BRAF_F1:
5' CCTCACAGTAAAAATAGGTGATTTTGGTCT 3'
配列番号4 BRAF_R5:
5' TAGCCTCAATTCTTACCATCCACAAAA 3'
Claims (11)
- B-Rafキナーゼ阻害剤に対する癌細胞の感度を決定する方法であって:
−癌を有する患者から得られた癌細胞に由来する核酸試料中の標的ポリヌクレオチド配列を、当該標的ポリヌクレオチド配列を増幅するプライマー対を用いて増幅すること、ここで増幅は、BRAF中のV600E、V600D又はV600Kのいずれかの突然変異配列の存在を検出する、配列番号1に記載されている配列から成る標識オリゴヌクレオチドプローブの存在下実施される;及び
−B-RafにおけるV600E、V600D又はV600Kのいずれかの突然変異の有無を検出し、それにより当該患者のB-Raf阻害剤に対する癌の感度を決定すること;
を含んで成り、V600E、V600D又はV600Kのいずれかの突然変異の存在が、当該患者のB-Raf阻害剤に対する癌の感度を決定する、方法。 - 増幅が、V600部位における野生型配列の存在を検出する第二プローブの存在下実施される、請求項1に記載の方法。
- 前記第二プローブが、配列番号2に記載のヌクレオチド配列のうちの少なくとも15個連続のヌクレオチドを含んで成る、請求項2に記載の方法。
- 前記プライマー対のプライマーの一方が、配列番号3に記載のヌクレオチド配列のうちの少なくとも15個連続のヌクレオチドを含んで成る、請求項1に記載の方法。
- 前記プライマー対のプライマーの一方が、配列番号4に記載のヌクレオチド配列のうちの少なくとも15個連続のヌクレオチドを含んで成る、請求項1に記載の方法。
- B-Raf阻害剤を用いた治療の候補者である患者を検出するためのキットであって、BRAF中のV600E、V600D又はV600Kのいずれかの突然変異を検出し、そして配列番号1に記載のヌクレオチド配列を含んで成る、第一対立遺伝子特異的プローブを含んで成る、キット。
- 野生型BRAF配列を検出し、そして配列番号2に記載のヌクレオチド配列のうちの少なくとも15個連続のヌクレオチド配列を含んで成る第二対立遺伝子特異的プローブを更に含んで成る、請求項6に記載のキット。
- V600部位を含んで成るBRAFの標的領域を増幅するプライマー対を更に含んで成る、請求項6に記載のキット。
- 前記プライマー対が、配列番号3に記載のヌクレオチド配列のうちの少なくとも15個連続のヌクレオチドを含んで成るプライマーを含んで成る、請求項8に記載のキット。
- 前記プライマー対が、配列番号3及び配列番号4に記載の配列を有するプライマーを含んで成る、請求項8に記載のキット。
- 前記第一プローブが、配列番号1に記載のヌクレオチド配列を有しており、そして前記キットが、配列番号2に記載のヌクレオチド配列を有する第二プローブを更に含んで成り、そしてプライマー対が、配列番号3及び配列番号4に記載のヌクレオチド配列を有するプライマーを含んで成る、請求項6に記載のキット。
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