JP2018517129A - 前立腺がんの分析のための組成物及び方法 - Google Patents
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Abstract
【選択図】なし
Description
本出願は、2015年4月21日出願の米国仮出願第62/150724号の優先権の利益を主張し、この出願はその全体が参照により本明細書に援用される。
本明細書において交換可能に使用される用語「腫瘍」又は「がん」は、腫瘍性又は悪性の細胞の成長、増殖、又は転移を特徴とするいずれかの医学的状態を指し、固形がん及び白血病などの非固形がんの両方を含む。
本明細書に開示される方法は、侵襲的、外科的生検を行うことなく、STEAP1−ADC療法の恩恵を受けるであろう患者を同定することができ、且つ疾患の進行とSTEAP1−ADC療法の有効性を監視することができるという未だ満たされていない需要を満たす。この方法は、侵襲性で痛みを伴う生検を行うことなく、STEAP1−ADC療法のよい候補となる患者及びSTEAP1−ADC療法に応答するであろう患者の検出及び選択を可能にする。またこの方法は、STEAP1−ADCによる治療の間に、STEAP1+CTC及びSTEAP1の発現を頻繁に監視することを可能にする。頻繁な監視により、外科的生検ではその侵襲性により可能でない、用量調節及び個別化が可能となる。したがって、本明細書に開示される方法は、ADC治療法が特異的バイオマーカーに焦点を当てたものであり、その特異的バイオマーカーのレベルを、ADC療法の前、最中及び後に監視及び検出することができると有利であるため、ADC療法のために特に重要である。
別の態様では、本発明は、抗体15A5が結合するのと実質的に同じエピトープに結合する抗体を提供し、抗体15A5は、微生物寄託番号PTA−12259を有するハイブリドーマ細胞により生成される。
抗体は、例えば米国特許第4816567号に記載されているような組み換え方法及び組成物を用いて生成することができる。一実施態様では、本明細書に記載される抗STEAP−1抗体をコードする単離された核酸が提供される。このような核酸は、抗体のVLを含むアミノ酸配列、及び/又は抗体のVHを含むアミノ酸配列(例えば、抗体の軽鎖及び/又は重鎖)をコードしうる。さらなる実施態様では、そのような核酸を含む一又は複数のベクター(例えば、発現ベクター)が提供される。さらなる実施態様では、そのような核酸を含む宿主細胞が提供される。このような一実施態様では、宿主細胞は:(1)抗体のVLを含むアミノ酸配列及び抗体のVHを含むアミノ酸配列をコードする核酸を含むベクター、又は(2)抗体のVLを含むアミノ酸配列をコードする核酸を含む第一ベクター及び抗体のVHを含むアミノ酸配列をコードする核酸を含む第ニベクターを含む(例えば、同ベクターで形質転換されている)。一実施態様において、宿主細胞は、真核生物細胞、例えばチャイニーズハムスター卵巣(CHO)細胞、又はリンパ系細胞(例えば、Y0、NS0、Sp20細胞)である。一実施態様において、上述のように、抗体の発現に適した条件下で、抗体をコードする核酸を含む宿主細胞を培養することと、任意選択的に宿主細胞(又は宿主細胞培地)から抗体を回収することとを含む、抗STEAP−1抗体を作製する方法が提供される。
本明細書において提供される抗体は、前立腺がんの診断試薬の製造に使用することができる。抗体は、診断目的に適した検出可能標識とさらにコンジュゲートし、適切な形態、例えば凍結乾燥させた粉末で、又は適切な溶液の形態で提供される。
本発明の別の態様では、前立腺ガンの診断又は予後予測に有用な組成物を含むテストキットが提供される。
以下は、本発明の方法及び組成物の実施例である。上に提供された一般的な説明を前提として、他の様々な実施態様が実施されうることが理解される。
この実施例に示されるように、免疫蛍光に基づくアッセイは、CTCの表面上におけるSTEAP1の頑強な検出と、治療後のCTCの総数並びにSTEAP1+CTCの減少とを実証することができた。
進行性mCRPCを有する60名の患者は、0.3から2.8mg/kgの範囲のSTEAP1−ADCを、三週に一度投与された。ADC活性を、PSAの減少により、>6ヶ月の研究時間において測定した。≧2mg/kgの用量において、≧50%のPSAの減少が、10/45名(22%±12.1)の患者に観察された。ADC活性は、IHC 3+腫瘍を有する患者において最も顕著であった。≧2mg/kgにおいて、20/45名の患者(44%±14.5)が、ベースラインにおいて7.5mL当たり≧5という望ましくないCTC数を有しており、評価可能と考慮された。1回目の治療サイクル後、望ましくない数から望ましい数へのCTC変換が11/20名の患者(55%±21.8)に見られ、PSA減少と有意に相関していた(スピアマンの順位r=0.74)。CTCは、FACSにより、14/29の試料(49%±31−66%)において、EpCAM+/CD45−イベントに容易に検出可能なSTEAP1+シグナルを示した。STEAP1+イベントは、0〜74%の範囲のEpCAM+/CD45−特性を示し、FACSでソートされたEpCAM+/CD45−イベントは、前立腺がんの推定マーカーAR及びKLK3に沿ってSTEAP1+RT−PCR発現を示した。EPICにより、STEAP1は18/42の試料(43%±14.98)に検出され、範囲はSTEAP1+CTCの1〜56%であった。治療後、ベースラインと比較して、STEAP1+の患者の割合の減少と、STEAP1=CTCの増加が観察された。17名の患者がSTEAP1 PET試験に参加した。STEAP1−ADCで治療した14名の患者のうち、4/14名(29%±12〜55%)の患者においてPSAに≧50%の減少があった。治療時間は骨転移SUVmaxと相関していた(r=0.63)。高いSUV転移の8のPET誘導生検すべてでSTEAP1 IHC 2+/3+が確認された。
STEAP1シグナルを、SKBR3(STEAP1ネガティブコントロール)及びLnCaP(STEAP1ポジティブコントロール)前立腺がん細胞株において、増幅プロトコールの存在下及び非存在下において測定した(図9)。STEAP+試料のカットオフはそれぞれ、非増幅プロトコールを用いた場合>3のシグナル、増幅プロトコールを用いた場合>8のシグナルであった。非増幅プロトコールは、LnCaP(ポジティブコントロール)細胞株にSKBR3(ネガティブコントロール)細胞株の7.6倍のシグナル増加を示した。増幅プロトコールは、LnCaP(ポジティブコントロール)細胞株にSKBR3(ネガティブコントロール)細胞株の25倍のシグナル増加を示した(図9A)。STEAP1シグナルを増幅した結果、LnCaP(ポジティブコントロール)細胞株の平均STEAP1シグナルが8.1倍に増加したが、背景(即ち、SKBR3ネガティブコントロールシグナル)の増加はわずか2.4倍であった(図9A)。
血液試料を、STEAP1−ADCによる治療の前、最中、及び後に採取した。図10に示すように、治療が進行するにつれ、STEAP1+CTCの数は減少した。
Claims (20)
- CTCを用いて被験者の前立腺がんを診断するための方法であって:
a)被験者から採取された血液試料由来のCTCを同定すること;
b)CTCを、STEAP1に特異的に結合する抗体、CD45に特異的に結合する抗体、及びサイトケラチンに特異的に結合する抗体と接触させること;
c)CTCをDAPIで染色すること;並びに
d)DAPI染色及び結合した抗体を、シグナルを強化するためのチラミドシグナル増幅(TSA)プロトコールを用いて検出すること
を含み、サイトケラチン及び前立腺特異的マーカーを発現し、DAPI染色を呈し、且つCD45を発現しないCTCの存在により、被験者が前立腺がんを有することが予測される、方法。 - 抗STEAP1抗体が、微生物寄託番号PTA−12259を有するハイブリドーマ細胞により生成される15A5抗体である、請求項1に記載の方法。
- がん細胞上のSTEAP1の発現レベルを決定することをさらに含む、請求項1又は2に記載の方法。
- STEAP1の発現レベルに基づいてがん細胞をグレード付けすること、及び各グレードのがん細胞のパーセンテージを決定することをさらに含む、請求項1から3のいずれか一項に記載の方法。
- 各グレードのグレードスコアを、前記グレードのがん細胞のパーセンテージに、前記グレードの前立腺特異的マーカーの発現レベルを表す固有のグレード数を乗じることにより計算すること、及びすべてのグレードスコアを合算してHスコアを取得することをさらに含み、Hスコアにより被験者の前立腺がんのステージが示される、請求項1から4のいずれか一項に記載の方法。
- がん細胞が、上皮由来のがん細胞に特異的に結合するリガンドを含む捕獲組成物を用いて血液試料から同定される、請求項1から5のいずれか一項に記載の方法。
- リガンドが、がん細胞上に優勢に発現される上皮抗原に特異的に結合する抗体である、請求項6に記載の方法。
- 上皮抗原が上皮細胞接着分子(EpCAM)である、請求項7に記載の方法。
- 同定されたがん細胞が、血液試料から分離された細胞分画に富んでいる、請求項6から8のいずれか一項に記載の方法。
- 細胞分画が磁場の下で分離される、請求項9に記載の方法。
- 捕獲組成物中のリガンドが、磁性粒子(例えば、コロイド状磁性粒子、例えば、コロイド状磁性ナノ粒子)に結合する、請求項10に記載の方法。
- リガンドがEpCAM抗体を含む、請求項11に記載の方法。
- 抗STEAP−1抗体が、≦1000nMのKDでSTEAP−1に結合する、請求項1から12のいずれか一項に記載の方法。
- 抗STEAP−1抗体、抗CD45抗体、及び/又は抗サイトケラチン抗体が、検出可能な標識にコンジュゲートされる、請求項1から13のいずれか一項に記載の方法。
- 前立腺がんに罹患している被験者におけるSTEAP1−ADC療法の有効性を予測する方法であって:
a)被験者から採取された血液試料由来のCTCを同定すること;
b)CTCを、STEAP1に特異的に結合する抗体、CD45に特異的に結合する抗体、及びサイトケラチンに特異的に結合する抗体と接触させること;
c)CTCをDAPIで染色すること;並びに
d)DAPI染色及び結合した抗体を、シグナルを強化するためのチラミドシグナル増幅(TSA)プロトコールを用いて検出すること
を含み、サイトケラチン及びSTEAP1を発現し、DAPI染色を呈し、且つCD45を発現しないCTCの存在により、STEAP1−ADCの有効性が予測される、方法。 - 前立腺がんに罹患している被験者におけるSTEAP1−ADC療法への応答を、シグナルを強化するためのチラミドシグナル増幅(TSA)プロトコールを用いて監視する方法であって:
a)第1群のCTCを、STEAP1に特異的に結合する抗体、CD45に特異的に結合する抗体、及びサイトケラチンに特異的に結合する抗体と接触させること並びに細胞をDAPIで染色することであって、第1群のがん細胞が、STEAP1−ADCによる治療前に被験者から採取された第1の血液試料に由来するものである、こと;
b)STEAP1及びサイトケラチンを発現し、DAPIで染色される第1群中のCTCの数を決定すること;
c)第2群のCTCを、STEAP1に特異的に結合する抗体、CD45に特異的に結合する抗体、及びサイトケラチンに特異的に結合する抗体と接触させること並びに細胞をDAPIで染色することであって、第2群のがん細胞が、STEAP1−ADCによる治療の間に又は後で被験者から採取された第2の血液試料に由来するものである、こと;
d)STEAP1及びサイトケラチンを発現し、DAPIで染色される第2群中のがん細胞の量を決定すること;
e)b)で決定されたSTEAP1及びサイトケラチンを発現し、且つDAPIで染色されるCTCの量を、d)で決定されたものと比較すること
を含み、CTCの量の減少及び/又はSTEAP1+CTCの減少により、被験者における前立腺がん治療法への応答が示される、方法。 - STEAP1−ADCが、細胞傷害性剤に共有結合する抗STEAP1抗体を含む、請求項1から16のいずれか一項に記載の方法。
- 細胞傷害性剤が、毒素、化学療法剤、薬物部分、モノメチルアウリスタチンE(MMAE)、抗生物質、放射性同位体、及び核酸分解酵素から選択される、請求項17に記載の方法。
- DAPI、STEAP1、CD45、及びサイトケラチンのシグナルが、FACSを用いて検出される、請求項1から18のいずれか一項に記載の方法。
- DAPI、STEAP1、CD45、及びサイトケラチンのシグナルが、免疫組織化学を用いて検出される、請求項1から18のいずれか一項に記載の方法。
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |