JP6702991B2 - T細胞免疫サブセットを検出するためのアッセイ及びその使用の方法 - Google Patents
T細胞免疫サブセットを検出するためのアッセイ及びその使用の方法 Download PDFInfo
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Description
本出願は、その全体が参照により本明細書に組み込まれる、2014年11月3日出願の米国仮特許出願第62/074,594号に基づく優先権を主張する。
ASCIIテキストファイルでの以下の提出物の内容は、参照によりその全体が本明細書に組み込まれる。コンピュータ可読形態(CRF)の配列表(ファイル名:146392029040SEQLIST.TXT、記録日:2015年10月28日、サイズ:185KB)。
本明細書において記載されるか、または参照される技法及び手順は、当業者によって一般に十分に理解され、従来の方法論、例えば、Sambrook et al.,Molecular Cloning:A Laboratory Manual 3d edition(2001)Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.、Current Protocols in Molecular Biology(F.M.Ausubel,et al.eds.,(2003))、the series Methods in Enzymology(Academic Press,Inc.):PCR2:A Practical Approach(M.J.MacPherson,B.D.Hames and G.R.Taylor eds.(1995)),Harlow and Lane,eds.(1988)Antibodies,A Laboratory Manual,and Animal Cell Culture(R.I.Freshney,ed.(1987))、Oligonucleotide Synthesis(M.J.Gait,ed.,1984)、Methods in Molecular Biology,Humana Press、Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,1998)Academic Press、Animal Cell Culture(R.I.Freshney),ed.,1987)、Introduction to Cell and Tissue Culture(J.P.Mather and P.E.Roberts,1998)Plenum Press、Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,1993−8)J.Wiley and Sons、Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.);Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987)、PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994)、Current Protocols in Immunology(J.E.Coligan et al.,eds.,1991)、Short Protocols in Molecular Biology(Wiley and Sons,1999)、Immunobiology(C.A.Janeway and P.Travers,1997)、Antibodies(P.Finch,1997)、Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988−1989)、Monoclonal Antibodies:A Practical Approach(P.Shepherd and C.Dean,eds.,Oxford University Press,2000)、Using Antibodies:A Laboratory Manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999)、The Antibodies(M.Zanetti and J.D.Capra,eds.,Harwood Academic Publishers,1995)、及びCancer:Principles and Practice of Oncology(V.T.DeVita et al.,eds.,J.B.Lippincott Company,1993)に記載される、広く利用されている方法論等を使用して、一般的に用いられる。
本発明を詳細に説明する前に、本発明は特定の組成物または生物系に限定されず、組成物及び生物系は当然ながら様々であり得ることを理解されたい。本明細書に使用される用語は特定の実施形態を説明することのみを目的とするものであり、限定的であるよう意図されるものではないことも理解されたい。
(a)アミノ酸残基26〜32(L1)、50〜52(L2)、91〜96(L3)、26〜32(H1)、53〜55(H2)、及び96〜101(H3)で発生する超可変ループ(Chothia and Lesk,J.Mol.Biol.196:901−917(1987))、
(b)アミノ酸残基24〜34(L1)、50〜56(L2)、89〜97(L3)、31〜35b(H1)、50〜65(H2)、及び95〜102(H3)で発生するCDR(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD(1991))、
(c)アミノ酸残基27c〜36(L1)、46〜55(L2)、89〜96(L3)、30〜35b(H1)、47〜58(H2)、及び93〜101(H3)で発生する抗原接触体(MacCallum et al.J.Mol.Biol.262:732−745(1996))、ならびに
(d)HVRアミノ酸残基46〜56(L2)、47〜56(L2)、48〜56(L2)、49〜56(L2)、26〜35(H1)、26〜35b(H1)、49〜65(H2)、93〜102(H3)、及び94〜102(H3)を含む、(a)、(b)、及び/または(c)の組み合わせを含む。
100×分数X/Y
式中、Xは、配列アライメントプログラムALIGN−2によって、そのプログラムのA及びBのアライメントにおいて完全な一致としてスコア化されたアミノ酸残基の数であり、Yは、B中のアミノ酸残基の総数である。アミノ酸配列Aの長さがアミノ酸配列Bの長さと等しくない場合、Bに対するAのアミノ酸配列同一性%は、Aに対するBのアミノ酸配列同一性%と等しくはならないことが理解されよう。特に別途定めのない限り、本明細書で使用される全てのアミノ酸配列同一性%値は、直前の段落に記載されるように、ALIGN−2コンピュータプログラムを使用して得られる。
EVQLVESGGGLVQPGGSLRLSCAAS(配列番号214)−H1−WVRQAPGKGLEWV(配列番号215)−H2−RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(配列番号216)−H3−WGQGTLVTVSS(配列番号217)の各々の少なくとも一部または全てを含む。
がんを有する対象のOX40アゴニスト治療薬に対する応答性を予測する方法が、本明細書に提供される。これらの方法は、がんを有する対象から得られたがん細胞及びリンパ球を含有する試料中のCD4+ OX40+ Foxp3+リンパ球の数が、対象をOX40アゴニスト治療薬に対して応答性であるかまたは非応答性であるかで分類するために使用され得るという本明細書に記載される発見に部分的に基づく。がんを有する対象から得られたがん細胞及びリンパ球を含有する試料中のCD4+ OX40+ Foxp3+リンパ球の数を使用して、対象の予後を判定し得る、かつ/または本開示のOX40アゴニストで治療する対象を選択し得ることも、本開示のさらなる発見である。がんを有する対象から得られた転移性がん細胞及びリンパ球を含有する試料中のCD4+リンパ球、OX40+リンパ球、CD4+ OX40+ Foxp3+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数を使用して、対象の予後を判定し得る、かつ/または本開示のOX40アゴニストで治療する対象を選択し得ることも、本開示のさらなる発見である。さらに、本明細書に記載されるように、OX40アゴニスト抗体は、対象から得られたがん細胞(原発性がん細胞及び転移性がん細胞を含む)ならびにリンパ球を含む試料中のOX40+リンパ球、CD4+ OX40+ Foxp3+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球を定量化する方法において、用途をさらに見出し得る。
及び/またはDIVMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(配列番号184)の配列を含む軽鎖を含む。いくつかの実施形態では、抗体は、少なくとも1つ、2つ、3つ、4つ、5つ、または6つのUS7550140に記載されるような抗体008の超可変領域(HVR)配列を含む。いくつかの実施形態では、抗体は、US7550140に記載されるような抗体008の重鎖可変領域配列及び/または軽鎖可変領域配列を含む。
本明細書に記載の方法のために有用なOX40アゴニストは、抗体に限定するものであるとは決して企図されていない。非抗体OX40アゴニストが企図され、当該技術分野では既知である。
ある特定の実施形態では、本明細書に提供される抗体は、≦1μM、≦100nM、≦10nM、≦1nM、≦0.1nM、≦0.01nM、または≦0.001nM(例えば10−8M以下、例えば10−8M〜10−13M、例えば10−9M〜10−13M)の解離定数(Kd)を有する。
ある特定の実施形態では、本明細書に提供される抗体は、抗体断片である。抗体断片には、Fab、Fab’、Fab’−SH、F(ab’)2、Fv、及びscFv断片、ならびに以下に記載される他の断片が含まれるが、これらに限定されない。ある特定の抗体断片の概説については、Hudson et al.Nat.Med.9:129−134(2003)を参照されたい。scFv断片の概説に関して、例えばPluckthun,in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.,(Springer−Verlag,New York),pp.269−315(1994)を参照されたく、WO93/16185ならびに米国特許第5,571,894号及び同第5,587,458号もまた参照されたい。エピトープ残基を結合し、増加したインビボ半減期を有するサルベージ受容体を含むFab及びF(ab’)2断片の考察に関しては、米国特許第5,869,046号を参照されたい。
ある特定の実施形態では、本明細書に提供される抗体は、キメラ抗体である。特定のキメラ抗体は、例えば、米国特許第4,816,567号、及びMorrison et al.,Proc.Natl.Acad.Sci.USA,81:6851−6855(1984))に記載されている。一例において、キメラ抗体は、非ヒト可変領域(例えば、マウス、ラット、ハムスター、ウサギ、またはサル等の非ヒト霊長類に由来する可変領域)及びヒト定常領域を含む。さらなる例において、キメラ抗体は、クラスまたはサブクラスが親抗体のそれから変更された、「クラススイッチされた」抗体である。キメラ抗体には、それらの抗原結合断片が含まれる。
ある特定の実施形態では、本明細書に提供される抗体は、ヒト抗体である。ヒト抗体は、当該技術分野で既知の様々な技法を使用して産生することができる。ヒト抗体は、概して、van Dijk and van de Winkel,Curr.Opin.Pharmacol.5:368−74(2001)及びLonberg,Curr.Opin.Immunol.20:450−459(2008)に記載されている。
本発明の抗体は、コンビナトリアルライブラリを、所望の活性(単数または複数)を有する抗体についてスクリーニングすることによって、単離されてもよい。例えば、ファージディスプレイライブラリを生成し、かかるライブラリを、所望の結合特性を保有する抗体についてスクリーニングするための、多様な方法が当該技術分野で知られている。かかる方法は、例えば、Hoogenboom et al.in Methods in Molecular Biology178:1−37(O’Brien et al.,ed.,Human Press,Totowa,NJ,2001)に概説され、例えば、McCafferty et al.,Nature348:552−554、Clackson et al.,Nature,352:624−628(1991)、Marks et al.,J.Mol.Biol.222:581−597(1992)、Marks and Bradbury,in Methods in Molecular Biology248:161−175(Lo,ed.,Human Press,Totowa,NJ,2003)、Sidhu et al.,J.Mol.Biol.338(2):299−310(2004)、Lee et al.,J.Mol.Biol.340(5):1073−1093(2004)、Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467−12472(2004)、及びLee et al.,J.Immunol.Methods 284(1−2):119−132(2004)にさらに記載されている。
ある特定の実施形態では、本明細書に提供される抗体は、多重特異性抗体、例えば、二重特異性抗体である。多重特異性抗体は、少なくとも2つの異なる部位に対する結合特異性を有するモノクローナル抗体である。ある特定の実施形態では、結合特異性の一方は、OX40に対するものであり、他方は、任意の他の抗原に対するものである。ある特定の実施形態では、二重特異性抗体は、OX40の2つの異なるエピトープに結合し得る。二重特異性抗体をまた使用して、細胞傷害性薬剤を、OX40を発現する細胞に限局させてもよい。二重特異性抗体は、完全長抗体または抗体断片として調製することができる。
ある特定の実施形態では、本明細書に提供される抗体のアミノ酸配列変異型が企図される。例えば、抗体の結合親和性及び/または他の生物学的特性を向上させることが望ましい場合がある。抗体のアミノ酸配列変異型は、抗体をコードするヌクレオチド配列中に適切な修飾を導入することによって、またはペプチド合成によって、調製されてもよい。かかる修飾には、例えば、抗体のアミノ酸配列からの残基の欠失、及び/またはそこへの残基の挿入、及び/またはその中での残基の置換が含まれる。最終構築物に到達するように、欠失、挿入、及び置換を任意で組み合わせることができるが、但し、その最終構築物が、所望の特性、例えば、抗原結合性を保有することを条件とする。
ある特定の実施形態では、1つ以上のアミノ酸置換を有する抗体変異型が提供される。置換型変異誘発に対する目的の部位には、HVR及びFRが含まれる。保存的置換は、表Aにおいて、「好ましい置換」の見出しの下に示される。より実質的な変化は、表Aにおいて、「例示的な置換」の見出しの下に提供され、またアミノ酸側鎖クラスを参照して以下にさらに記載される。アミノ酸置換が目的の抗体中に導入され、その産物が、所望の活性、例えば、保持された/向上した抗原結合、減少した免疫原性、または向上したADCC若しくはCDCについて、スクリーニングされてもよい。
(1)疎水性:ノルロイシン、Met、Ala、Val、Leu、Ile;
(2)中性親水性:Cys、Ser、Thr、Asn、Gln;
(3)酸性:Asp、Glu;
(4)塩基性:His、Lys、Arg;
(5)鎖の配向に影響を及ぼす残基:Gly、Pro;
(6)芳香族:Trp、Tyr、Phe。
ある特定の実施形態では、本明細書に提供される抗体は、抗体がグリコシル化される程度を増加または減少させるように改変される。抗体へのグリコシル化部位の付加または欠失は、1つ以上のグリコシル化部位が作り出されるか、または除去されるように、アミノ酸配列を改変することによって、簡便に遂行され得る。
ある特定の実施形態では、1つ以上のアミノ酸修飾が、本明細書に提供される抗体のFc領域に導入され、それによりFc領域変異型を生成することができる。Fc領域変異型は、1つ以上のアミノ酸位置にアミノ酸修飾(例えば、置換)を含む、ヒトFc領域配列(例えば、ヒトIgG1、IgG2、IgG3、またはIgG4 Fc領域)を含んでもよい。
ある特定の実施形態では、抗体の1つ以上の残基がシステイン残基で置換されている、システイン操作された抗体、例えば、「チオマブ(thioMAb)」を作り出すことが望ましい場合がある。具体的な実施形態では、置換残基は、抗体の利用しやすい部位において生じる。これらの残基をシステインで置換することによって、反応性のチオール基がそれにより抗体の利用しやすい部位に位置付けられ、それを使用して、薬物部分またはリンカー−薬物部分等の他の部分に抗体を複合して、本明細書にさらに記載される免疫複合体を作り出してもよい。ある特定の実施形態では、以下の残基、軽鎖のV205(Kabat付番)、重鎖のA118(EU付番)、及び重鎖Fc領域のS400(EU付番)のうちの任意の1つ以上が、システインで置換されてもよい。システイン操作された抗体は、例えば、米国特許第7,521,541号に記載されるように生成され得る。
ある特定の実施形態では、本明細書に提供される抗体は、当該技術分野で既知であり、かつ容易に入手可能な、追加の非タンパク質性部分を含有するようにさらに修飾されてもよい。抗体の誘導体化に好適な部分には、水溶性ポリマーが含まれるが、これに限定されない。水溶性ポリマーの非限定的な例としては、ポリエチレングリコール(PEG)、エチレングリコール/プロピレングリコールのコポリマー、カルボキシメチルセルロース、デキストラン、ポリビニルアルコール、ポリビニルピロリドン、ポリ−1,3−ジオキソラン、ポリ−1,3,6−トリオキサン、エチレン/無水マレイン酸コポリマー、ポリアミノ酸(ホモポリマーまたはランダムコポリマーのいずれか)、及びデキストランまたはポリ(n−ビニルピロリドン)ポリエチレングリコール、プロプロピレン(propropylene)グリコールホモポリマー、プロリプロピレン(prolypropylene)オキシド/エチレンオキシドコポリマー、ポリオキシエチル化ポリオール(例えば、グリセロール)、ポリビニルアルコール、ならびにそれらの混合物が挙げられるが、これらに限定されない。ポリエチレングリコールプロピオンアルデヒドは、水中でのその安定性により、製造における利点を有し得る。ポリマーは、任意の分子量のものであってもよく、分岐していても非分岐であってもよい。抗体に結合したポリマーの数は様々であり得、1つを超えるポリマーが結合される場合、それらは、同じ分子または異なる分子であり得る。一般に、誘導体化に使用されるポリマーの数及び/または種類は、向上させるべき抗体の特定の特性または機能、抗体誘導体が規定の条件下で療法において使用されるかどうか等を含むがこれらに限定されない検討事項に基づいて決定することができる。
抗体は、例えば、米国特許第4,816,567号に記載されるもの等の組み換え方法及び組成物を使用して産生され得る。一実施形態では、本明細書に記載される抗OX40抗体をコードする単離核酸が提供される。かかる核酸は、抗体のVLを含むアミノ酸配列及び/またはVHを含むアミノ酸配列(例えば、抗体の軽鎖及び/または重鎖)をコードし得る。さらなる実施形態では、かかる核酸を含む1つ以上のベクター(例えば、発現ベクター)が提供される。さらなる実施形態では、かかる核酸を含む宿主細胞が提供される。1つのかかる実施形態では、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、または(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1のベクター、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2のベクターを含む(例えば、それらで形質転換されている)。一実施形態では、宿主細胞は、真核性、例えば、チャイニーズハムスター卵巣(CHO)細胞またはリンパ系細胞(例えば、Y0、NS0、Sp20細胞)である。一実施形態では、抗OX40抗体を作製する方法が提供され、本方法は、上記抗体をコードする核酸を含む宿主細胞を、抗体の発現に好適な条件下で培養することと、任意で、抗体を宿主細胞(または宿主細胞培養培地)から回収することとを含む。
本明細書に提供される抗OX40抗体は、それらの物理的/化学的特性及び/または生物活性について、当該技術分野で既知の様々なアッセイによって、特定され、スクリーニングされ、または特徴付けられてもよい。
一態様において、本発明の抗体は、例えば、ELISA、ウェスタンブロット等の既知の方法によって、その抗原結合活性について試験される。OX40結合性は、当該技術分野で既知の方法を使用して決定されてもよく、例示的な方法が本明細書に開示される。一実施形態では、結合性は、放射免疫測定法を使用して測定される。例示的な放射免疫測定法は、実施例において例証される。OX40抗体をヨウ素化し、一定濃度のヨウ素化抗体及び減少した濃度の連続希釈された非標識OZ X40抗体を含有する競合反応混合物を調製する。OX40を発現する細胞(例えば、ヒトOX40で安定的にトランスフェクトされたBT474細胞)を、反応混合物に付加する。インキュベーションに続いて、細胞を洗浄し、遊離ヨウ素化OX40抗体を、細胞に結合したOX40抗体から分離する。例えば、細胞に関連する放射能を計数することによって結合したヨウ素化OX40抗体のレベルが決定され、標準的な方法を使用して結合親和性が決定される。別の実施形態では、OX40抗体の表面発現OX40に結合する(例えば、T細胞サブセット上で)能力を、フローサイトメトリーを使用して評価する。末梢血白血球が得られ(例えば、ヒト、カニクイザル、ラット、またはマウスから)、細胞は血清で遮断される。標識OX40抗体を連続希釈物内に付加し、T細胞サブセットを特定する(当該技術分野で既知の方法を使用して)ためにT細胞もまた染色する。試料のインキュベーション及び洗浄に続いて、フローサイトメーターを使用して細胞を分類し、当該技術分野で周知の方法を使用してデータを分析する。別の実施形態では、表面プラズモン共鳴を使用してOX40結合性を分析してもよい。例示的な表面プラズモン共鳴方法は、実施例において例証される。
一態様において、生物活性を有する抗OX40抗体を特定するためのアッセイが提供される。生物活性には、例えば、OX40を結合すること(例えば、ヒト及び/またはカニクイザルOX40を結合する)、OX40媒介シグナル伝達(例えば、NFkB媒介性転写)を増加させること、ヒトOX40を発現する細胞(例えば、T細胞)を枯渇させること、ADCC及び/または食作用によってヒトOX40を発現する細胞を枯渇させること、例えばエフェクターT細胞増殖を増加させる及び/またはエフェクターT細胞によるサイトカイン産生(例えば、ガンマインターフェロン)を増加させることでTエフェクター細胞機能(例えば、CD4+エフェクターT細胞)を増強すること、例えばメモリーT細胞増殖を増加させる及び/またはメモリーT細胞によるサイトカイン産生(例えば、ガンマインターフェロン)を増加させることでメモリーT細胞機能(例えば、CD4+メモリーT細胞)を増強すること、制御性T細胞機能を阻害すること(例えば、エフェクターT細胞機能(例えば、CD4+エフェクターT細胞機能)のTreg抑制を減少させることによって)、ヒトエフェクター細胞を結合することが含まれる。インビボ及び/またはインビトロでかかる生物活性を有する抗体もまた提供される。
本発明はまた、化学療法剤または化学療法薬、成長阻害剤、毒素(例えば、タンパク質毒素、細菌真菌、植物、若しくは動物起源の酵素活性毒素、またはそれらの断片)、若しくは放射性同位体等の1つ以上の細胞傷害性薬剤と複合された本明細書における抗OX40抗体を含む、免疫複合体を提供する。
対象から得られたがん細胞及びリンパ球を含有する試料中のCD4+ OX40+ Foxp3+リンパ球の数を測定し、参照と比較して、試料中のCD4+ OX40+ Foxp3+リンパ球の数に基づいて、対象の予後を判定することによってがんを有する対象の予後を判定する方法が本明細書に提供され、試料中のCD4+ OX40+ Foxp3+リンパ球の数の増加は、対象が予後の改善を有し得ることを示す。これらの方法は、腫瘍試料におけるCD4+ OX40+ Foxp3+リンパ球(すなわちOX40+ Treg細胞)の発生率の増加が、患者の予後の改善と相関するという、本明細書に記載される発見に部分的に基づく。対象から得られた転移性がん細胞及びリンパ球を含有する試料中のCD4+リンパ球、OX40+リンパ球、CD4+ OX40+ Foxp3+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数を測定し、参照と比較して、試料中のOX40+リンパ球、CD4+ OX40+ Foxp3+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数に基づいて対象の予後を判定することによってがんを有する対象の予後を判定する方法が本明細書にさらに提供され、試料中のOX40+リンパ球、CD4+ OX40+ Foxp3+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数の増加は、対象が予後の改善を有し得ることを示す。これらの方法は、腫瘍試料におけるCD4+リンパ球、全OX40+リンパ球、CD4+ OX40+ Foxp3+リンパ球(すなわちOX40+ Treg細胞)、またはCD4+ OX40+ Foxp3−リンパ球(すなわちOX40+ Teff細胞)の発生率の増加が、患者の予後の改善と相関し、かつこれらのリンパ球サブタイプの各々の発生率が、対象からの一致した原発性腫瘍及び転移性試料において強く相関するという、本明細書に記載される発見に部分的に基づく。
一態様において、個体のがんを治療する、またはその進行を遅延させる方法であって、個体に有効量のOX40アゴニストを投与することを含む方法が、本明細書に提供される。本開示の方法は、とりわけ、がんまたはT細胞機能障害性疾患の治療のための腫瘍免疫原性の増加等、免疫原性の増強が所望される病態を治療する際に、用途を見出し得る。これらの方法によって、多様ながんが治療され得るか、または、それらの進行が遅延され得る。
上述の方法、及び本明細書において説明される方法で用いるために、製造者のキットまたは物品も提供される。かかるキットは、対象からのがん細胞及びリンパ球を含む試料中の、CD4+リンパ球、OX40+リンパ球、CD4+OX40+Foxp3+リンパ球、またはCD4+OX40+Foxp3−リンパ球を検出するのに特異的な少なくとも1つの試薬を含んでもよい。いくつかの実施形態では、製造者のキットまたは物品は、ヒトCD4(例えば、NeoMarkersクローン4B12)に結合する抗体、ヒトOX40(例えば、抗体1A7)に結合する抗体、及び/またはFoxP3(例えば、Abcamクローン236A/E7)に結合する抗体を含む。いくつかの実施形態では、製造者のキットまたは物品は、本明細書に記載されるOX40アゴニスト(例えば、抗OX40アゴニスト抗体)をさらに含む。いくつかの実施形態では、製造者のキットまたは物品は、注入用静菌水(BWFI)、リン酸緩衝生理食塩水、リンガー溶液、及びデキストロース溶液等の他の試薬及び/または緩衝剤をさらに含む。製品は、他の緩衝液、希釈剤、フィルタ、針、及びシリンジを含む、商業的立場及びユーザの立場から望ましい他の物質をさらに含んでもよい。試薬及び抗体は、容器内にあってもよい。好適な容器としては、例えば、ボトル、バイアル、シリンジ、IV溶液バッグなどが挙げられる。容器は、ガラスまたはプラスチックなど、様々な材料から形成されていてもよい。いくつかの実施形態では、製造者のキット及び物品は、本明細書に記載される方法で試薬及び/または抗体を使用するための指示を提供する、(容器上または容器と関連し得る)ラベルまたは添付文書をさらに含む。
結腸直腸がん(CRC)患者の腫瘍微小環境におけるOX40+T細胞の数の増加は、転帰の改善と関連付けられている(Petty,J.K.,et al.(2002)Am.J.Surg.183(5):512−8)。しかしながら、OX40+T細胞の集団は、不均一であり、数ある中でも、CD4+Foxp3+制御性T細胞(Treg)及びCD4+Foxp3−エフェクターT細胞(Teff)を含む。
症例選択
原発部位(n=48)及び一致した転移(n=19)を含むホルマリン固定パラフィン包埋(FFPE)CRC標本を、治療歴及び生存転帰で注釈を付けられた集合から含めた。患者の年齢は、26〜85歳の範囲に亘り、第I病期(4)、第II病期(12)、第III病期(17)、及び第IV病期(8)の疾患が、示された。症例に、高分化腺がんまたは中分化腺がん(n=36)を、低分化(n=4)または粘液特徴(8)のいずれかを有する追加の症例と共に、含めた。
逐次的アプローチを、二重及び三重の免疫蛍光染色を含む全ての免疫標識に関して追跡調査した。CD4(NeoMarkersクローン4B12)、FoxP3(Abcamクローン236A/E7)、及びOX40(クローン1A7)抗体を、使用した。
画像を、100倍の最終倍率で、標準蛍光フィルタを使用して、Ariol SL−50自動スライド走査プラットフォーム(Leica Microsystems,Buffalo Grove,Il.)によって得た。全体のタイル状の画像をエクスポートし、Matlabソフトウェアパッケージ(バージョンR2012b,Mathworks,Natick,MA)で分析した。
診断日からあらゆる原因で死亡した日までの全生存期間を計算し、患者が依然として生存している日を、患者が最後に生存していたことが分かっている日で打ち切った。(中央値で二分した)腫瘍関連T細胞サブセットのレベルと全生存期間との間の関連性を、対数順位検定を使用して評価した。腫瘍関連T細胞サブセットと診断時のTNM病期との間の関連性を評価するために、クラスカル・ワリス検定を採用した。多重検定補正を適用しなかった。
原発部位(n=48)及び一致した転移(n=19)を含む、48人のCRC患者からの腫瘍を染色し、デジタルで撮像した。
上述のOX40+ T細胞サブセットとがんの病期との間に認められた相関のために、次にこれらのT細胞サブセットの存在を、予後に関して分析した。
腫瘍試料中に認められたOX40+細胞と腫瘍病期と患者生存との間に認められた相関を考えて、次に、OX40+ T細胞サブセットを、原発性腫瘍試料と転移性腫瘍試料とのペアにおいて分析した。
Claims (32)
- がんを有する対象の予後を判定するための方法であって、
(a)前記対象から得られたがん細胞及びリンパ球を含む試料中のCD4+ OX40+ Foxp3+リンパ球の数を測定することと、
(b)参照と比較して、前記試料中のCD4+ OX40+ Foxp3+リンパ球の数に基づいて前記対象の前記予後を判定することと、を含み、前記試料中のCD4+ OX40+ Foxp3+リンパ球の数の増加は、前記対象が予後の改善を有し得ることを示す、前記方法。 - 前記予後の改善が、全生存期間の増加を含む、請求項1に記載の方法。
- 前記予後の改善が、無憎悪生存期間の増加を含む、請求項1または請求項2に記載の方法。
- CD4+ OX40+ Foxp3+リンパ球の数が、前記対象からの前記試料の目的の異なる領域内のCD4+ OX40+ Foxp3+リンパ球の中央値、平均値、または平均数である、請求項1〜3のいずれか一項に記載の方法。
- CD4+ OX40+ Foxp3+リンパ球の数が、前記試料の目的の前記領域内の全細胞に対して正規化される、請求項4に記載の方法。
- 前記参照が、前記対象の前記がんと同じ種類及び/または病期を有するがんから得られたがん細胞及びリンパ球を含む試料中のCD4+ OX40+ Foxp3+リンパ球の数に基づく、請求項1〜5のいずれか一項に記載の方法。
- 前記参照が、前記対象の前記がんと同じ種類及び/または病期を有するがんから得られた試料中のCD4+ OX40+ Foxp3+リンパ球の中央値、平均値、または平均数である、請求項6に記載の方法。
- 前記試料中のCD4+ OX40+ Foxp3+リンパ球の数の測定が、
(a)前記試料中のCD4の発現を示すリンパ球を標識することと、
(b)ステップ(a)の後に、前記試料中のOX40の発現を示すリンパ球を標識することと、
(c)ステップ(b)の後に、前記試料中のFoxp3の発現を示すリンパ球を標識することと、
(d)ステップ(c)の後に、前記試料中のCD4+ OX40+ Foxp3+リンパ球の数を測定することと、を含む、請求項1〜7のいずれか一項に記載の方法。 - ステップ(a)とステップ(b)との間、及び/またはステップ(b)とステップ(c)との間に、EDTA緩衝液またはクエン酸緩衝液を含む溶液で前記試料を処理することをさらに含む、請求項8に記載の方法。
- CD4の発現、OX40の発現、及びFoxp3の発現を示すリンパ球が、免疫蛍光染色によって標識される、請求項8または9に記載の方法。
- OX40の発現を示すリンパ球が、(a)配列番号2のアミノ酸配列を含むHVR−H1と、(b)配列番号3のアミノ酸配列を含むHVR−H2と、(c)配列番号4のアミノ酸配列を含むHVR−H3と、(d)配列番号5のアミノ酸配列を含むHVR−L1と、(e)配列番号6のアミノ酸配列を含むHVR−L2と、(f)配列番号7、26、または27から選択されるアミノ酸配列を含むHVR−L3と、を含む抗体を使用して標識される、請求項10に記載の方法。
- 前記試料中のCD4+ OX40+ Foxp3+リンパ球の数を定量化することが、前記免疫蛍光染色を撮像することを含む、請求項10または11に記載の方法。
- 前記がんが、結腸直腸がん、非小細胞肺がん、腎細胞がん腫、膀胱がん、卵巣がん、膠芽腫、神経芽腫、黒色腫、乳がん、胃がん、及び肝細胞がんからなる群から選択される、請求項1〜12のいずれか一項に記載の方法。
- 前記乳がんが、トリプルネガティブ乳がんである、請求項13に記載の方法。
- 前記試料が、がん細胞及びリンパ球を含み、前記がん細胞が、原発性腫瘍から、または転移した癌腫からのものである、請求項1〜14のいずれか一項に記載の方法。
- 前記CD4+ OX40+ Foxp3+リンパ球が、腫瘍浸潤性リンパ球である、請求項1〜15のいずれか一項に記載の方法。
- がんを有する対象の予後を判定するための方法であって、
(a)前記対象から得られた転移性がん細胞及びリンパ球を含む試料中のCD4+ OX40+ Foxp3+リンパ球の数を測定することと、
(b)参照と比較して、前記試料中のCD4+ OX40+ Foxp3+リンパ球の数に基づいて前記対象の前記予後を判定することと、を含み、前記試料中のCD4+ OX40+ Foxp3+リンパ球の数の増加は、前記対象が予後の改善を有し得ることを示す、前記方法。 - 前記予後の改善が、全生存期間、および/又は無憎悪生存期間の増加を含む請求項17に記載の方法。
- CD4+ OX40+ Foxp3+リンパ球の数が、前記対象からの前記試料の目的の異なる領域内のCD4+ OX40+ Foxp3+リンパ球の中央値、平均値、または平均数である、請求項17または18に記載の方法。
- CD4+ OX40+ Foxp3+リンパ球の数が、前記試料中の前記領域内の全細胞に対して正規化される、請求項19に記載の方法。
- 前記参照が、前記対象の前記がんと同じ種類及び/または病期を有するがんから得られた転移性がん細胞及びリンパ球を含む試料中のCD4+ OX40+ Foxp3+リンパ球の数に基づく、請求項17〜20のいずれか一項に記載の方法。
- 前記参照が、前記対象の前記がんと同じ種類及び/または病期を有するがんから得られた試料中のCD4+ OX40+ Foxp3+リンパ球の中央値、平均値、または平均数である、請求項21に記載の方法。
- 前記がんが、結腸直腸がん、非小細胞肺がん、腎細胞がん腫、膀胱がん、卵巣がん、膠芽腫、神経芽腫、黒色腫、乳がん、胃がん、及び肝細胞がんからなる群から選択される、請求項17〜22のいずれか一項に記載の方法。
- 前記乳がんが、トリプルネガティブ乳がんである、請求項23に記載の方法。
- 前記CD4+ OX40+ Foxp3+リンパ球が、腫瘍浸潤性リンパ球である、請求項17〜24のいずれか一項に記載の方法。
- トリプルネガティブ乳がんを有する対象の予後を判定するための方法であって、
(a)前記対象から得られた転移性トリプルネガティブ乳がん細胞及びリンパ球を含む試料中のOX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数を測定することと、
(b)参照と比較して、前記試料中のOX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数に基づいて前記対象の前記予後を判定することと、を含み、前記試料中のOX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数の増加は、前記対象が予後の改善を有し得ることを示す、前記方法。 - 前記予後の改善が、全生存期間、および/又は無憎悪生存期間の増加を含む、請求項26に記載の方法。
- OX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数が、前記対象からの前記試料の目的の異なる領域内のOX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の中央値、平均値、または平均数である、請求項26又は27に記載の方法。
- OX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数が、前記試料中の前記領域内の全細胞に対して正規化される、請求項28に記載の方法。
- 前記参照が、前記対象の前記トリプルネガティブ乳がんと同じ種類及び/または病期を有するトリプルネガティブ乳がんから得られた転移性トリプルネガティブ乳がん細胞及びリンパ球を含む試料中のOX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の数に基づく、請求項26〜29のいずれか一項に記載の方法。
- 前記参照が、前記対象の前記トリプルネガティブ乳がんと同じ病期を有するトリプルネガティブ乳がんから得られた試料中のOX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球の中央値、平均値、または平均数である、請求項30に記載の方法。
- 前記OX40+リンパ球、またはCD4+ OX40+ Foxp3−リンパ球が、腫瘍浸潤性リンパ球である、請求項26〜31のいずれか一項に記載の方法。
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- 2015-11-02 WO PCT/US2015/058674 patent/WO2016073378A1/en active Application Filing
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US20180238878A1 (en) | 2018-08-23 |
BR112017009151A2 (pt) | 2018-03-06 |
MX2017005750A (es) | 2017-12-15 |
US20160161485A1 (en) | 2016-06-09 |
KR20170086540A (ko) | 2017-07-26 |
WO2016073378A1 (en) | 2016-05-12 |
CN106796235A (zh) | 2017-05-31 |
SG11201703448QA (en) | 2017-05-30 |
IL251977A0 (en) | 2017-06-29 |
EP3215850B1 (en) | 2019-07-03 |
RU2017119009A3 (ja) | 2019-06-14 |
US10845364B2 (en) | 2020-11-24 |
EP3215850A1 (en) | 2017-09-13 |
CA2966523A1 (en) | 2016-05-12 |
AR102517A1 (es) | 2017-03-08 |
CN106796235B (zh) | 2021-01-29 |
JP2018504610A (ja) | 2018-02-15 |
US20210109099A1 (en) | 2021-04-15 |
AU2015343337A1 (en) | 2017-06-15 |
RU2017119009A (ru) | 2018-12-05 |
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