JP4971788B2 - 細胞外Hsp90阻害剤 - Google Patents
細胞外Hsp90阻害剤 Download PDFInfo
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- JP4971788B2 JP4971788B2 JP2006504609A JP2006504609A JP4971788B2 JP 4971788 B2 JP4971788 B2 JP 4971788B2 JP 2006504609 A JP2006504609 A JP 2006504609A JP 2006504609 A JP2006504609 A JP 2006504609A JP 4971788 B2 JP4971788 B2 JP 4971788B2
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- cells
- hsp90
- aldu
- cell
- antibody
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Description
悪性腫瘍は、細胞を離脱し、新しい組織へ移動して、二次腫瘍を生じる。二次腫瘍を発生させる過程は、転移と呼ばれ、腫瘍細胞が原発腫瘍から離れた部位にコロニーを形成する複雑な過程である。Liotta((1986) Cancer Res. 46, 1-7)は、転移の過程について3段階仮説を提案した:第一段階は、細胞表面受容体を介した腫瘍細胞接着である。固着した腫瘍細胞は、次に、加水分解性酵素を分泌する、または酵素を分泌するように宿主細胞を誘導し、マトリックスを局所的に分解することができる。マトリックス溶解は、腫瘍細胞表面に近い非常に限局化された領域に起こる可能性が最も高い。第三段階は、タンパク質分解により改変されたマトリックスの領域への腫瘍細胞移動運動である。このように、マトリックスの浸潤は、単に受動的増殖圧力によるだけでなく、能動的な生化学的機構を必要とする。周囲の正常な組織の分解は、悪性腫瘍の浸潤性の主眼点である。転移形成の過程は、腫瘍細胞の浸潤性に依存する。それゆえに、浸潤性を阻害し、それを以て、原発腫瘍の転移を防ぐ薬剤を開発することが有用であると思われる。
本発明は、細胞外Hsp90に特異的に結合することができる分子に関する。さらに、本発明の分子は、望ましい場合には、検出可能な群で標識されうる、または生物結合体の部分でありうる。
本明細書に用いられる場合、他に規定がない限り、以下の定義が適用されるものとする。
本明細書に記載された発明がより完全に理解されうるために、以下の詳細な説明が提供される。
a)細胞へのHsp90阻害剤の取り込みを本質的に妨げる条件下で、細胞を1つまたは複数のHsp90阻害剤に接触させる段階;
b)段階a)に従って処理された細胞の移動を分析する段階;
c)段階a)に従って処理された細胞の移動を、処理されていない細胞と比較する段階、および任意で
d)段階a)に従って処理された細胞の移動のパーセンテージを、処理されていない細胞に対して決定する段階。
a)増幅可能なリガンドディスプレイユニット(amplifiable ligand-displaying unit)(ALDU)のライブラリーを癌細胞、例えば肉腫細胞に接触させる段階;
b)その癌細胞およびそれに結合したALDUを、その癌細胞に結合していないALDUから分離する段階;
c)その癌細胞に結合したALDUを増幅する段階;
d)ALDU、または段階c)後のALDUに由来する、Hsp90のその生物活性に影響を及ぼすリガンドを、機能スクリーニングアッセイにより同定する段階;
e)ALDU、または段階d)後のALDUに由来する、Hsp90に結合する能力があるリガンドを、同定する段階;および
f)リガンドの化学的独自性(identity)を測定する段階。
e)ALDU、例えば、単離されたファージ、を組換えHsp90に接触させる段階;
f)そのHsp90を緩衝界面活性剤および/または高塩溶液で洗浄する段階;ならびに
g)ALDU、例えばHsp90に結合したファージ、を溶離する段階;ならびに
h)リガンド、例えばその溶離されたALDU、例えばファージ、により示される抗体または抗体断片の独自性を決定する段階。
実施例1:モノクローナル抗体(例えば、mAb 1.5.1)の作製
マウスを、3ヶ月に渡って、2回、固定または溶解のいずれかのHT-1080細胞で免疫した。固定については、集密的な(1x107個/75cm2フラスコ)HT-1080細胞を、PBSで1回、洗浄し、その後、PBSおよび掻爬で取り出した。細胞をピペッティングにより再懸濁し、220Xgで5分間、遠心分離し、10 mlの2% パラホルムアルデヒドで、4℃、10分間、固定した。細胞をPBSで洗浄し、その後、1 MLのPBSに再懸濁した。溶解された細胞は、集密的なHT-1080細胞をトリプシン処理し、PBSで1回、洗浄し、その後、12000xgで1分間、ペレット化することにより作製された。その後、細胞を、室温で5分間、溶解緩衝液(0.33 M トリス-HCl、pH 7.5における0.67 トリトン-X-100)中で溶解させた。その後、可溶化液を12000xgで5分間、遠心分離した。可溶化液をPD-10カラムに添加し、3.5 mL PBSでタンパク質を溶出させることにより、トリトン-X-100を除去した。タンパク質を、Micro-BCAキット(Pierce)を用いて定量し、150 μg/mL/マウスを免疫化に用いた。
2匹のBALB/cマウスをそれぞれ、2 x 107個のパラホルムアルデヒド固定化HT-1080細胞(ヒト線維肉腫細胞系;ATCC、CCL-121)で皮内に免疫化した。最初の免疫化後、39日の期間において2回、注射を繰り返し、マウスを屠殺し、脾臓を摘出して、液体窒素中に凍結させた。
一本鎖Fvは、固定HT-1080細胞で免疫化されたマウスから作製されたファージディスプレイライブラリーから選択された。ライブラリーは、ファージディスプレイベクターpXP10を用いて作製された。
スクリーニングのために、ファージディスプレイベクターに含まれる、選択されたscFvをコードする遺伝子を、発現ベクターpXP14に再クローニングした。このベクターは、StrepタグおよびEタグと融合したscFvの発現を方向づけ、繊維状ファージ遺伝子-3を含まない。単一コロニー由来の発現ベクター含有大腸菌TG1を、各ウェルが1つのscFvクローンのみを含むようにマイクロタイタープレートの個々のウェルにおいて増殖させた。細菌を96ウェルマイクロタイタープレート(#9297, TPP)での100 μg/ml アンピシリンおよび0.1% グルコースを追加した2xTYにおいて、0.7のOD600まで、30℃で増殖させた。発現を、0.5 mMの最終濃度でのIPTGで誘発し、25℃で一晩、継続させた。一本鎖Fv含有透明可溶化物を、25℃で1時間の50 μg/mlの最終濃度までの鶏卵リソチーム(#L-6876, Sigma)の添加および3000 x gで15分間の遠心分離により調製した。スクリーニングELISAの前に、透明可溶化物を、1時間のDMEM+10% FCSの等量の添加によりブロッキングした。スクリーニングELISAのために、HT-1080細胞を0.05% EDTAで収集し、パラホルムアルデヒドで固定し、PBSにおいて1x107細胞/mlまで希釈し、96ウェルUV架橋プレート(Corning Costar)のウェル上へ固定化した。UV架橋プレートのウェルをMPBSでブロッキングし、scFv含有のブロッキングされた透明可溶化物を25℃で1.5時間、加えた。プレートをPBS+0.1% ツイーン-20で2回、およびPBSで1回、洗浄し、HRP結合α-Eタグ(#27-9413-01, Pharmacia Biotech;0.1% ツイーン-20でMPBSに1:5000に希釈)と1時間、インキュベートし、PBS+0.1% ツイーン-20で3回、およびPBSで3回、洗浄し、POD(#1 484 281, Roche)で発色させて、シグナルを370 nmで読んだ。
scFv1およびそれの遺伝子のシーケンシングは、プライマーpXP2 Seq2
およびpXP2 Seq1
を用いて、Sequiserve GmbH, Vaterstetten, Germanyにより行われた。アミノ酸配列およびヌクレオチド配列は図に示されている。
精製された抗HT-1080scFvの標的細胞へ特異的に結合する能力を試験するために、本発明者らは、HT-1080細胞(ATCC CCL-121)および対照細胞系としてHs-27細胞(106細胞/ml)を用いて蛍光活性化セルソーター(FACS)分析を行った(図参照)。細胞をCellWash(BD(Becton, Dickinson and Company) #349524)において4℃で20分間、純粋なscFvの10 μg/mlとインキュベートし、洗浄し、結合したscFvを、二次のFITC標識抗Eタグモノクローナル抗体(Amersham #27-9412-01))で検出した。試料を洗浄し、Becton Dickinson FACSscan上で分析した。図8は、scFv1と反応する細胞について、対数蛍光強度(FL1-H;x軸)対相対的細胞数(カウント;y軸)を示している。細い線は、対照細胞系(HS-27)を、太い線は、HT-1080細胞を表している。scFv1は、対照細胞系と比較して10倍まで高いシグナルをもって腫瘍細胞系を特異的に染色している。
scFvを以下の方法によりフルオレセインイソチオシアネート(FITC)(Molecular Probes, Eugene, USA #F1906)で標識した:乾燥ジメチルスルホキシドにおけるFITCの10 mg/ml溶液のアリコートを、PBS/0.5 M NaHCO3、pH 9.5に溶解されたscFv1の100 μgに、30:1(FITC:scFv1)の比率において添加した。試料を室温で撹拌しながら2時間、インキュベートし、遊離FITCを脱塩カラム(2 Micro Spin G-25, Pharmacia 27-5325-01)を用いて分離した。標識の比率を質量分析法およびUV/VIS分光法により測定し、タンパク質濃度を280 nmで、FITC濃度を494 nmで計算した。
新鮮に作製された乾燥ジメチルスルホキシドにおける10 mg/mL FITCの溶液に、精製されたmAb 1.5.1抗体(T-gel Absorbant, Pierce Biotechnology #20500)を1:5の分配で添加した。0.5 M NaHCO3、pH 9.5の等量をその反応物に添加し、反応物を室温で2時間、揺り動かしながらインキュベートした。遊離FITCを脱塩カラム(PD-10, Amersham #17-0851-01)を用いて分離した。比率は、494 nMにおけるVIS分光法によりFITC濃度を計算し、その標識からの完全なタンパク質回収を仮定することにより、決定された。
ChemoTx(登録商標)システム(Neuro Probe Inc. #106-8, Gaithersburg)が、8 μm フィルタートラックをエッチングされたポリカーボネート細孔サイズ、5.7 mm直径/部位である96ウェル型での使い捨ての走化性/細胞遊走チャンバーとして用いられる。
この実施例は、FITC標識scFvの使用(標識については実施例7を参照)および浸潤アッセイの間のCALI過程の組み込みを除いて、一般的には、実施例8と同一である。
ChemoTx(登録商標)システム(Neuro Probe Inc. #106-8, Gaithersburg)が、8 μm フィルタートラックをエッチングされたポリカーボネート細孔サイズ、5.7 mm直径/部位である96ウェル型での使い捨ての走化性/細胞遊走チャンバーとして用いられた。
96ウェルプレート(TPP #9296)(細胞培養処理された)は、列B〜Hにおいて、ダルベッコのPBS(Gibco #14040-091)におけるコラーゲンS I型 1 μg/ウェル(Roche #10982929)でコーティングされ、列Aウェル10〜12においては、2% BSA(Sigma #A-7030)/ダルベッコのPBSで、4℃で一晩、コーティングされた。プレートをダルベッコのPBSで洗浄し、列B〜Hおよび列Aウェル10〜12を2% BSA/ダルベッコのPBSで、37℃で1時間、ブロッキングし、ダルベッコのPBSで再び、洗浄した。HT-1080細胞を、10% FCS(Gibco #10270106)を含むグルタマックスI(862 mg/l (Gibco #31966-021)を追加したDMEMにおいて70〜80%集密まで増殖させた。細胞を、DMEM/グルタマックスI/0.1% BSA(Sigma #A-7030)で2回、洗浄し、その後、ビスベンズイミドH 33342(Sigma #B-2261)でインサイチューで標識し、DMEM/グルタマックスI/0.1% BSAにおいて、37℃、7.5% CO2で15分間、1:100に希釈した。細胞を、DMEM/グルタマックスI/0.1% BSAで2回、洗浄し、回収のために、DMEM/グルタマックスI/0.1% BSAを37℃、7.5% CO2で15分間、負荷した。PBS w/o Ca2+、Mg2+(Gibco, 10010-015)での2回の洗浄後、細胞を、0.5 mM EDTA(Sigma #E8008)で脱離させ、ダルベッコのPBS/0.1% BSA/10 mM ヘペス(Gibco #15630-056)で収集し、ダルベッコのPBS/0.1% BSA/10 mM ヘペスで2回、洗浄し、ダルベッコのPBS/0.1% BSA/10 mM ヘペスに懸濁し、ダルベッコのPBS/0.1% BSA/10 mM ヘペスで6.7 x 106細胞/mlまで希釈した。6.7 x 106細胞/mlを、CALI後の接着の阻害についての対照としてFITC標識抗ベータ1 インテグリンモノクローナル抗体(JB1, Chemicon #MAB1963)の40 μg/mlと、およびHT-1080特異的FITC標識scFvと、1:1で、氷上で1時間、インキュベートした。1.3 x 105個のHT-1080細胞/ウェルまたはHT-1080細胞/scFvもしくはモノクローナル抗体の希釈物を、2つの96ウェルプレートの、黒色の、超薄型透明平底のオプティックス(Costar #3615)に3連でピペットで移した。一方のプレートを暗闇に氷上で保持したが、他方のプレートには488 nmでの連続波レーザーを氷塊上で照射した(0.5 W、30秒)。DMEM/グルタマックスI/0.1% BSAでの6.7 x 105細胞/mlへの希釈後、HT-1080細胞およびHT-1080細胞/scFvの希釈物を、コーティングおよびブロッキングされたプレート上へ3連(照射された3連に加えて非照射の3連)でピペットで移した。列Aウェル10〜12において、DMEM/グルタマックスI/0.1% BSAを含む6.7 x 105細胞/mlをバックグラウンド対照としてピペットで移した。プレートを37℃、7.5% CO2で1時間、インキュベートし、ダルベッコのPBSで2回、洗浄し、非接着細胞を洗い流した。列Aウェル1〜9において、1 x 104細胞/ウェルから4 x 104細胞/ウェルまでの標準曲線を行い、すべての他のウェルにおいては、50 μl ダルベッコのPBSをピペットで移した。コラーゲンS I型に接着した(標準曲線の場合は、接着しなかった)細胞の蛍光を、370/460 nmの励起/発光波長を用いてFluostar Galaxy (bMG)マイクロプレートリーダー上で測定した。scFv1は、コラーゲンS I型へのHT-1080細胞の接着を50%、阻害した(データ示されず)。
mAbの免疫沈降のために、HT-1080細胞の集密単層を、プロテアーゼ阻害剤カクテル(Boehringer Mannheim)を含む1 mM トリス-Cl、pH 8.0を用いて、4℃で15分間、溶解させた。細胞を、ダンス型ホモジナイザーを用いて5分間、さらに溶解させた。可溶化物を、抗マウスIgG-アガロース(Sigma)に4℃で1時間、あらかじめ吸着させた。標的ハイブリドーマクローン(10 μL/1 mg 抽出された細胞)由来の腹水を4℃で一晩、インキュベートした。抗体-タンパク質複合体を、抗マウスIgG-アガロースビーズを用いて、4℃で1時間、単離した。免疫沈降されたタンパク質をSDS-PAGEおよびクーマシー染色により分析し、mAb免疫沈降物を標的腹水での免疫ブロットによりさらに分析した。
mAb標的同定のために、SDS-PAGEにより分離された免疫沈降物からのクーマシー染色バンドを次に、ゲル内消化にかけた。トリプシン処理されたペプチド断片を、75 μM ナノスプレーC18カラム(New Objectives)をもつSurveyor HPLC and LCQ Deca Ion Trap質量分析計(ThermoFinningan)上に走らせた。得られたPMF(ペプチド質量断片)を、NCBIおよびSwissProtデータベースにおいて、種ヒト(Homo sapiens)についてのすべてのエントリーを検索するために用いた。
表面タンパク質免疫細胞化学のために、HT-1080細胞の集密単層(96ウェルの透明薄型底プレートのウェルあたり40,000個)を、ハイブリドーマ上清と37℃/7% CO2で1時間、インキュベートした。3回の、5分間、リン酸緩衝食塩水(PBS)における0.1% ウシ血清アルブミン(BSA)の洗浄後、細胞を2% パラホルムアルデヒドにおいて2分間、固定した。0.1% BSA/PBSの洗浄のもう1ラウンドを行い、FITC-ウサギ-抗マウス二次抗体を室温で1時間、加えた。同じ洗浄を繰り返し、三次FITC-ヤギ-抗ウサギ抗体を室温で1時間、加えた。洗浄の最終ラウンド後、細胞を画像化まで4℃でPBS中に保存した。表面染色は、Neofluar 40X/0.75開口数レンズを用いるZeiss Axiovert 10における顕微鏡観察法により可視化された。画像は、FITCについて480での励起フィルターセットおよび535での発光フィルターセットを用いて収集された。結果は、図6、6.1および6.2に示されている。
細胞表面タンパク質のビオチン化は、Hanwell at al(J Biol Chem 277:9772)に記載されているように行われた。表面ビオチン化プールおよび非ビオチン化細胞内プールをSDS-PAGEゲル上で分離し、抗Hsp90(Stressgen #SPA-830)または抗α-アクチニン-4(Martin A. Pollak, Childrens Hospital, Boston, MA)での免疫ブロッティングのために転写した。結果は、図7に示されている。Hsp90は、表面(S)および細胞内(IC)の両方の局在性を示すが、α-アクチニンは、細胞内プールにおいてのみ見出される。mAb 1.5.1もまた、表面および細胞内局在性を示す(データ示されず)。
8 x 106個のHT-1080細胞/mLを示された濃度におけるノボビオシンまたはナリジクス酸で1時間、前処理し、浸潤アッセイを実施例9.1に記載されているように行った。ノボビオシンは、浸潤の用量依存性阻害を示す(>0.5 mMにおいてp<0.01)。ナリジクス酸は、浸潤に影響を及ぼさない。このアッセイにおけるノボビオシン処理された細胞の生存度は、影響を及ぼされなかった(データ示されず)。各データ点は、薬剤なしの対照に対して標準化され、3連での2つの実験の平均+s.e.m.を表している。
40,000個のHT-1080細胞を所定の濃度におけるノボビオシンまたはナリジクス酸で6時間、処理した。処理された細胞からの条件培地を、10 mg/mL ゼラチンを含むSDS-PAGEゲルを用いて分離した。SDSを、2.5% トリトン-X-100を用いて室温で30分間、ゲルから除去した。タンパク質を、消化緩衝液(0.1 M トリス-Cl2、pH 8.0、5 mM CaCl2、0.04% NaN3)において37℃で48時間、ゼラチンを消化するようにさせておいた。ゲルをクーマシー染色し、濃度測定のために乾燥させた。平行なゲルを、タンパク質の等しい負荷を示すために銀染色した(データ示されず)。MMP活性は、試験されたノボビオシンの最高濃度において>75%減少し、酵素活性の低下は、ナリジクス酸については見られなかった。結果は、図11に示されている。
この実施例は、HT-1080細胞の表面におけるHsp90機能の阻害を実証する。ノボビオシン、クマリン抗生物質でのHT-1080線維肉腫細胞の処理が、基底膜障壁を侵入する細胞の能力を低下させ、また、マトリックスメタロプロテアーゼ(MMP)活性/分泌を有意に低下させることが示された。実験は、短い時間尺度(6時間)で行われるため、細胞内Hsp90標的の有意な欠乏は起こりえない。
マトリックスメタロプロテイナーゼMMP2はHT-1080条件培地由来のHsp90αと免疫共沈降することが示されうる(図17)。その後、本発明者らは、Hsp90の阻害がMMP2活性を低下させるかどうかを試験するために、既知のHsp90阻害剤、ゲルダナマイシン、をHT-1080細胞へ適用した。HT-1080細胞を無血清培地において20 μM ゲルダナマイシン(GA)で処理し、分泌されたMMP2を酵素電気泳動法によりアッセイした。MMP2(72 kDa ゼラチナーゼ)活性は、GA処理後、〜35%(p<0.01)減少した。等しい負荷を保証するために、条件培地における総タンパク質含有量を銀染色により可視化した。(図18を参照)。
Claims (9)
- 細胞外Hsp90を発現する増殖性疾患、癌または転移を予防および/または処置する方法における使用のための、細胞外Hsp90に結合することができる抗体または抗体断片より選択される、ヒト細胞外Hsp90阻害剤。
- モノクローナル抗体である、請求項1記載の阻害剤。
- 検出可能な標識で標識されている、請求項1記載の阻害剤。
- 増殖性疾患、癌または転移を予防および/または処置する方法における使用のための、ヒト細胞外Hsp90阻害剤をコードする核酸分子であって、阻害剤は、ヒト細胞外Hsp90に結合することができる抗体または抗体の断片である、核酸分子。
- 請求項1〜3のいずれか一項記載の阻害剤および適した試験容器を含むキット。
- 阻害剤が標識されている、請求項5記載のキット。
- 以下の段階を含む、エキソビボで、細胞の浸潤および/もしくは転移挙動をスクリーニングまたは試験するための方法:
a)細胞へのHsp90阻害剤の取り込みを本質的に妨げる条件下で、細胞を1つまたは複数のヒトHsp90阻害剤に接触させる段階;
b)段階a)に従って処理された細胞の移動を分析する段階;
c)段階a)による細胞の移動を、処理されていない細胞と比較する段階、および任意で
d)処理されていない細胞と比較して移動のパーセンテージを決定する段階。 - 段階a)が、細胞の増殖に適した条件下で細胞をゲル様マトリックスに接触させる段階をさらに含み、かつ段階b)が、ゲル様マトリックスを通過する細胞の移動を分析する段階を含む、請求項7記載の方法。
- 増殖性疾患、癌または転移を予防および/または処置する方法において使用するための、請求項1〜3のいずれか一項記載の阻害剤を含む薬学的組成物。
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US7700819B2 (en) | 2001-02-16 | 2010-04-20 | Kci Licensing, Inc. | Biocompatible wound dressing |
US7959915B2 (en) * | 2003-03-12 | 2011-06-14 | Tufts University | Inhibitors of extracellular Hsp90 |
US9120774B2 (en) | 2004-11-03 | 2015-09-01 | University Of Kansas | Novobiocin analogues having modified sugar moieties |
CA2585091C (en) * | 2004-11-03 | 2016-07-19 | University Of Kansas | Novobiocin analogues as anticancer agents |
ES2399241T3 (es) | 2004-11-18 | 2013-03-26 | Synta Pharmaceuticals Corporation | Compuestos de triazol que modulan la actividad de HSP90 |
US7662813B2 (en) | 2005-08-18 | 2010-02-16 | Synta Pharmaceuticals Corp. | Triazole compounds that modulate HSP90 activity |
WO2008079914A1 (en) * | 2006-12-21 | 2008-07-03 | Novartis Ag | Antibody quantitation |
US20100111943A1 (en) * | 2007-03-22 | 2010-05-06 | Medical College Of Georgia Research Institute, Inc | Compositions and methods for inhibiting cancer metastasis |
AU2009208947B2 (en) | 2008-02-01 | 2014-02-06 | Takeda Pharmaceutical Company Limited | Oxim derivatives as HSP90 inhibitors |
WO2010001989A1 (ja) * | 2008-07-03 | 2010-01-07 | 協和発酵キリン株式会社 | 癌幹細胞及び/または癌前駆細胞の減少剤並びに癌の再発及び/または転移の予防剤 |
EP3121597A1 (en) * | 2008-08-18 | 2017-01-25 | Mesoblast, Inc. | Monoclonal antibody stro-4 |
CN101942017B (zh) | 2009-07-07 | 2013-08-14 | 清华大学 | 一种新的肿瘤标志物 |
CN103694332B (zh) * | 2009-07-07 | 2017-07-11 | 清华大学 | 一种新的肿瘤标志物 |
EP2560640A1 (en) | 2010-04-19 | 2013-02-27 | Synta Pharmaceuticals Corp. | Cancer therapy using a combination of a hsp90 inhibitory compounds and a egfr inhibitor |
KR102025142B1 (ko) | 2011-07-08 | 2019-09-26 | 슬로안-케테링인스티튜트퍼캔서리서치 | 표지된 hsp90 억제제의 용도 |
JP2014532712A (ja) | 2011-11-02 | 2014-12-08 | シンタ ファーマシューティカルズ コーポレーション | トポイソメラーゼi阻害剤とhsp90阻害剤の組合せを使用する癌療法 |
WO2013067165A1 (en) | 2011-11-02 | 2013-05-10 | Synta Pharmaceuticals Corp. | Combination therapy of hsp90 inhibitors with platinum-containing agents |
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