JP4511943B2 - Pd−1に対する抗体およびその使用 - Google Patents
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Description
本技術分野は、Programmed Death 1(PD−1)レセプターによって調節される免疫応答の調節に関する。
適応免疫応答は、T細胞およびB細胞と呼ばれるリンパ球の二種の主要なクラスの活性化、選択、およびクローン増殖を含む。抗原と接触した後、T細胞は増殖して抗原特異的エフェクター細胞へと分化し、一方でB細胞は増殖して抗体分泌細胞へと分化する。
本開示は、PD−1のアゴニストおよび/またはアンタゴニストとして作用し得る抗体を提供して、これにより、PD−1によって調節される免疫応答を調節する。本開示は、新規抗原結合フラグメントを含む抗PD−1抗体を、さらに提供する。本発明の抗PD−1抗体は、(a)PD−1(ヒトPD−1を含める)に特異的に結合すること;(b)PD−1とその天然リガンドとの相互作用をブロックすること;または(c)両方の機能を実行することが可能である。さらに、これらの抗体は、免疫調節性特性を有する(すなわち、これらの抗体は、免疫応答のPD−1関連ダウンレギュレーションの調節において有効であり得る)。使用方法および所望される効果に応じて、これらの抗体は、免疫応答を増強するか、または阻害するかのいずれかのために、使用され得る。
(定義)
用語「抗体」は、本開示において使用される場合、免疫グロブリンまたはそのフラグメント、もしくは誘導体を言い、そして、この抗体がインビトロまたはインビボで産生されるか否かにかかわらず、抗原結合部位を含む任意のポリペプチドを含む。この用語は、ポリクローナル抗体、モノクローナル抗体、モノ特異的抗体、ポリ特異的抗体、非特異的抗体、ヒト化抗体、一本鎖抗体、キメラ抗体、合成抗体、組換え抗体、ハイブリッド抗体、変異抗体、および移植抗体を含むが、これらに限定されない。本開示の目的のために、「インタクト抗体」のように、用語「インタクト」によって他に修飾されない限り、用語「抗体」はまた、抗体フラグメント(例えば、Fab、F(ab’)2、Fv、scFv、Fd、dAb)、ならびに抗原結合機能(すなわち、PD−1に特異的に結合する能力)を維持する他の抗体フラグメントを含む。代表的には、このようなフラグメントは、抗原結合ドメインを含む。
本開示は、新規抗原結合フラグメントを含む抗PD−1抗体を提供する。
本開示はまた、PD−1に特異的な抗体を得る方法を提供する。このような抗体のCDRは、表1に同定されるVHおよびVLの特異的な配列に限定されず、そしてPD−1に特異的に結合する能力を保持するこれらの配列の改変体を含み得る。このような改変体は、当該分野において周知の技術を用いて、当業者によって、表1に列挙される配列から誘導され得る。例えば、アミノ酸置換、アミノ酸欠失、またはアミノ酸付加は、FRにおいておよび/またはCDRにおいてなされ得る。FRにおける変更は、通常、抗体の安定性および免疫原性を向上するように設計され、一方でCDRにおける変更は、代表的に、この標的に対する抗体の親和性を上昇するように設計される。FRの改変体はまた、天然に存在する免疫グロブリンアロタイプ含む。このような、親和性を上昇させる変更は、CDRを変更させること、および抗体の標的に対する抗体の親和性を試験することを包含する慣例的な技術によって、経験的に決定され得る。例えば、保存的なアミノ酸置換は、任意の一つの開示されるCDR内になされ得る。種々の変更が、Antibody Engineering,第2版,Oxford University Press(編)Borrebaeck,1995に記載される方法に従って、なされ得る。これらは、配列内の機能的に等価なアミノ酸残基をコードする異なるコドンの置換によって変更されて、従って「サイレント」変化を生じるヌクレオチド配列を含むが、これらに限定されない。例えば、非極性アミノ酸としては、アラニン、ロイシン、イソロイシン、バリン、プロリン、フェニルアラニン、トリプトファン、およびメチオニンが挙げられる。極性中性アミノ酸としては、グリシン、セリン、スレオニン、システイン、チロシン、アスパラギン、およびグルタミンが挙げられる。陽性荷電(塩基性)アミノ酸としては、アルギニン、リジン、およびヒスチジンが挙げられる。陰性荷電(酸性)アミノ酸としては、アスパラギン酸およびグルタミン酸が挙げられる。この配列内のアミノ酸置換は、このアミノ酸が属するクラスの他のメンバーから選択され得る(表5を参照のこと)。さらに、このポリペプチドにおける任意のネイティブ残基はまた、アラニンで置換され得る(例えば、MacLennanら(1998)Acta Physio.Scand.Suppl.643:55−67;Sasakiら(1998)Adv.Biophys.35:1−24を参照のこと)。
(a)置換されるべきCDR3を含むか、またはCDR3のコード領域を欠いたかのいずれかのVHドメインをコードする核酸の開始レパートリーを提供する工程;
(b)このレパートリーを、VHのCDR3(すなわちH3)について、本明細書中に実質的に示されるようなアミノ酸配列をコードするドナー核酸と組み合わせて、このドナー核酸が、このレパートリーのCDR3領域内に挿入されることによって、VHドメインをコードする核酸の生成物レパートリーを提供する工程;
(c)この生成物レパートリーの核酸を発現する工程;
(d)PD−1に特異的な抗原結合フラグメントを選択する工程;ならびに
(e)この特異的抗原結合フラグメントまたはこの結合性フラグメントをコードする核酸を回収する工程。
本開示は、開示された抗体をコードする単離された核酸をさらに提供する。この核酸は、DNAまたはRNAを含み得、そして全体的、もしくは部分的に合成であり得るか、または組み換えであり得る。本明細書で示すような核酸配列への言及は、指定の配列を有するDNA分子を包含し、指定の配列を有するRNA分子を包含する。そのRNA配列においては、文脈が別なように必要としない限りは、UはTに置換されている。
開示された抗PD−1抗体は、免疫応答のPD−1関連ダウンレギュレーションを調節することが可能である。特定の実施形態において、免疫応答は、TcR/CD28に媒介される。この開示された抗体は、これらの使用の方法に依存して、PD−1のアゴニストまたはアンタゴニストのどちらかとして作用し得る。この抗体は、哺乳動物、特にヒトにおいて医学的障害を阻害、診断、または処置するために用いられ得る。本発明の抗体はまた、PD−1またはPD−1発現細胞を単離するためにも、用いられ得る。さらに、この抗体は、異常PD−1発現または機能に関連した障害の危険性を有するか、もしくは疑いのある被験体、または異常PD−1発現または機能に関連した障害を有する被験体を処置するために用いられ得る。
本開示は、抗PD−1抗体を含有する組成物を提供する。そのような組成物は、薬学的使用および患者への投与のために適し得る。代表的には、この組成物は、1つ以上の本発明の抗体および薬学的に受容可能な賦形剤を含む。語句「薬学的に受容可能な賦形剤」には、薬学的投与と両立するありとあらゆる溶媒、分散媒、コーティング、抗菌剤および抗真菌剤、等張剤、ならびに吸収遅延剤などが挙げられる。薬学的に活性な物質のための、このような媒体および薬剤の使用は、当該分野で周知である。その組成物はまた、補足された治療機能、追加された治療機能、または増強された治療機能を提供する他の活性な化合物をも含み得る。その薬学的な組成物はまた、コンテナ(container)、パック、またはディスペンサー(dispenser)中に投与のための指示書と一緒に含まれ得る。
scFvファージミドライブラリー(Vaughanら(Nature Biotech.(1996)14:309−314)により記載された1.38×1010ライブラリーの拡張版)を、ヒトPD−1に特異的な抗体を選択するために用いた。可溶PD−1融合タンパク質(20μg/ml(リン酸緩衝化生理食塩水(PBS)中))またはコントロール融合タンパク質(50μg/ml(PBS中))を、一晩4℃においてマイクロタイタープレートのウェルにコートした。ウェルをPBS中で洗浄し、MPBS(3%粉乳(PBS中))において1時間37℃でブロックした。精製ファージ(1012形質転換単位(tu))を、最終容量の100μlの3%MPBSにおいて1時間ブロックした。ブロックされたファージを、ブロックされたコントロール融合タンパク質のウェルに加え、1時間インキュベートした。次いで、ブロックされたファージおよび除外されたファージを、PD−1融合タンパク質でコートされたブロックされたウェルに移し、さらに1時間インキュベートした。ウェルをPBST(PBSコーティング 0.1%v/v Tween20)で5回洗浄し、次いでPBSで5回洗浄した。結合されたファージ粒子を溶出し、10mlの指数関数的に増殖するE.coli TG1に感染させるために用いた。感染細胞を、2TYブロス中で1時間37℃において増殖させ、次いで、2TYAGプレートに薄く塗り、一晩30℃においてインキュベートした。コロニーをプレートからはがして10mlの2TYブロスに移し、そして−70℃における貯蔵のための15%グリセロールを添加した。
PD−1についての抗体の特異性を決定するために、PD−1融合タンパク質およびコントロールタンパク質に対するファージELISAを行った。選択産出からの個々のE.coliコロニーを、1ウェルあたり100μlの2TYAG培地を含む96ウェルプレートに移した。M13K07ヘルパーファージを、10の感染多重度(moi)で指数関数的に増殖する培養物に加え、そのプレートをさらに1時間37℃においてインキュベートした。プレートをベンチトップ(benchtop)遠心分離器において、2000rpmにおいて10分間遠心分離した。その上清を除き、細胞ペレットを100μlの2TYAK中に再懸濁し、30℃において一晩、振盪しながらインキュベートした。次の日、プレートを2000rpmにおいて10分間遠心分離し、各ウェルからのファージ含有上清を新しい96ウェルプレートに移した。ファージサンプルを、ELISAの前に、3%MPBSの最終濃度においてブロックした。
PD−1結合scFv E.coliクローンを、2TYAGプレートに線引きし、一晩30℃にてインキュベートした。これらのプレートからのコロニーを、scFvクローン由来のVH領域およびVL領域を増幅するためpCANTAB6ベクター配列オリゴを用いて配列決定した。独特のPD−1結合クローンを、実施例4において記載の通りPD−1に対するPD−L1結合の中和について、アッセイした。scFvとIgGフォーマットとの間の配列の差は、scFvからIgGへの変換の間にPCRプライマーにより導入された変化に起因する。
scFv生成を、指数関数的に増殖する培養物に対して1mM IPTGを加え、一晩30℃にてインキュベーションすることによって誘導した。粗製のscFv含有の周辺質抽出物を、一晩の誘導からの細菌のペレットに浸透圧性ショックを受けさせることにより得た。ペレットを、20%(w/v)スクロース、50mM Tris−HCl、pH7.5、1mM EDTA中に再懸濁し、氷上で30分間冷やした。細胞の組織細片を、遠心分離によって除き、scFvをクロマトグラフィーおよびPBSへの緩衝液交換によって精製した。精製scFv(PD1−17、PD1−28、PD1−33、およびPD1−35)を、96ウェルマイクロタイタープレートアッセイにおいて、ビオチン標識されたヒトPD−L1融合タンパク質のプラスチック上に固定されたヒトPD−1融合タンパク質に対する結合を阻害する能力について試験した。ビオチン標識されたPD−L1融合タンパク質の結合を、AMDEX−アルカリホスファターゼを用いて検出し、生成されたシグナルを、マイクロタイタープレートリーダーを用いて405nmにおける吸光度を読み取ることにより、測定した。データは、全結合のパーセンテージとして表され、scFv濃度の力価を試験し、計算されたIC50値としてクローン潜在能(potency)を確立した。scFvおよびIgG抗体についてのクローン潜在能のデータを、表5に示す。
scFvクローン由来の重鎖および軽鎖V領域を、クローン特異的プライマーを用いたPCRによって増幅した。PCR産物を、適切な制限酵素を用いて消化し、ヒトIgG1重鎖定常領域(Takahashiら(1982)Cell 29,671)を含有するベクター、またはヒトλまたはκ軽鎖定常領域(Hieterら(1982)Nature 294,536)を含有するベクターにサブクローニングした。VHおよびVLセグメントの生殖系列に基づいて、κまたはλ軽鎖定常領域が変換のために用いられたかどうかを決定した(表7)。
阻害アッセイを実行し、抗体のPD−L1のPD−1に対する結合を遮断する能力を評価した。ELISAを、実施例2において記載された通りに、改変を含めて実行した。一次抗PD−1抗体と2時間室温においてインキュベーションした後、一定濃度(1μg/ml)のビオチン結合PD−L1−Igを加え、そのサンプルをさらに1時間RTにおいてインキュベートした。洗浄の後、飽和濃度のアビジンHRPを加え、1時間RTにおいてインキュベートした。結合していないアビジンHRPを、PBS/1%BSAを用いて洗浄した。このアッセイを、TBMを用いて現像した。
阻害アッセイを実行し、種々のヒト抗ヒトPD−1抗体によって認識される部位を位置づけた。ELISAを、実施例6に記載の通り、小さな改変を含めて実行した。一次抗体と2時間RTにおいてインキュベートした後、固定された濃度(0.25μg/ml)のビオチン結合PD−1抗体J110を加え、そのサンプルをさらに1時間RTにおいてインキュベートした。洗浄の後、飽和濃度のアビジンHRPを加え、1時間RTにおいてインキュベートした。結合していないアビジンHRPを、PBS/1%BSAを用いて洗浄した。このアッセイを、TBMを用いて現像した。
CD4+T細胞(5×104細胞/ウェル)を、抗hCD3+/−PD−L1−Fcまたは抗PD−1(PD1−17もしくはJ110)でコートされたトシルビーズ(Dynal,Great Neck,NY)を用いて刺激した。融合タンパク質の濃度または抗体力価を、図5のX軸に示した。72時間後、増殖を3Hチミジンの取り込みによって決定した。取り込まれた放射能を、LKB 1205プレートリーダーを用いて決定した。
増殖に関する可溶抗PD−1抗体の効果を評価するために、CD4+T細胞を、抗CD3/抗CD28コートのビーズを用いて、48時間プレ活性化し、収集し、そしてPD1−17、J110またはコントロールIgGの存在下において、指示された濃度のPHAプラス10ng/mlのIL−2を用いて再刺激した。抗体の各々を、種々の濃度において培養の開始時に加えた。増殖を、72時間において測定した。
PD−1により制御される免疫応答の調節は、免疫抑制効果または免疫応答の増強が所望される事例において有用である。この実施例は、PD−1アゴニストまたはPD−1アンタゴニストとしてのPD−1抗体の使用を記載し、それぞれ、疾患発症における被験体または確立された免疫障害もしくは癌を有する被験体を処置する。
Claims (30)
- 配列番号26(CDRL1)、配列番号27(CDRL2)および配列番号28(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖と、配列番号23(CDRH1)、配列番号24(CDRH2)および配列番号25(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖とを含む、抗PD−1抗体。
- 配列番号32(CDRL1)、配列番号33(CDRL2)および配列番号34(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖と、配列番号29(CDRH1)、配列番号30(CDRH2)および配列番号31(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖とを含む、抗PD−1抗体。
- 配列番号38(CDRL1)、配列番号39(CDRL2)および配列番号40(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖と、配列番号35(CDRH1)、配列番号36(CDRH2)および配列番号37(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖とを含む、抗PD−1抗体。
- 配列番号53(CDRL1)、配列番号54(CDRL2)および配列番号55(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖と、配列番号50(CDRH1)、配列番号51(CDRH2)および配列番号52(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖とを含む、抗PD−1抗体。
- 前記抗体がPD1−28である、請求項1に記載の抗PD−1抗体。
- 前記抗体がPD1−33である、請求項2に記載の抗PD−1抗体。
- 前記抗体がPD1−35である、請求項3に記載の抗PD−1抗体。
- 前記抗体がPD1−F2である、請求項4に記載の抗PD−1抗体。
- 前記抗体が、配列番号41および配列番号56からなる群から選択される少なくとも一つ配列の少なくとも100個の連続するアミノ酸のうちの任意の配列と、少なくとも95%同一であるアミノ酸配列に特異的に結合する、請求項1〜8のいずれか1項に記載の抗体。
- 前記抗体が、107M−1より大きい親和定数でPD−1の細胞外ドメインに特異的に結合する、請求項1〜8のいずれか1項に記載の抗体。
- 前記抗体が、10nM未満のIC50で、PD−LのPD−1への結合を阻害する、請求項9に記載の抗体。
- 前記抗体が、ヒト抗体である、請求項1〜8のいずれか1項に記載の抗体。
- 前記抗体が、IgG1またはIgG4である、請求項1〜8のいずれか1項に記載の抗体。
- 前記抗体が、IgG1λまたはIgG1κである、請求項13に記載の抗体。
- ヒトフレームワーク領域を含む請求項1〜8のいずれか1項に記載の抗体であって、該抗体は、PD−1とPD−L1との間の結合を阻害することが可能である、抗体。
- 請求項1〜8のいずれか1項に記載の抗体をコードする単離された核酸。
- 請求項16に記載の核酸を含む発現ベクター。
- 請求項17に記載のベクターを含む宿主細胞。
- 前記宿主細胞が、以下:E.Coli細菌、チャイニーズハムスター卵巣細胞、HeLa細胞、およびNS0細胞から選択される、請求項18に記載の宿主細胞。
- リンパ球と接触させるのに適した、請求項1〜8のいずれか1項に記載の抗PD−1抗体を含む、適応免疫応答を調節するための組成物。
- 前記リンパ球が、T細胞、B細胞、または単球である、請求項20に記載の組成物。
- 前記抗体が、支持マトリクス上で固定されるか、または架橋される、請求項20に記載の組成物。
- 前記支持マトリクスが、アガロース、デキストラン、セルロース、PVDF、シリカ、ナイロン、ダクロン、ポリスチレン、ポリアクリレート、ポリビニル、テフロン(登録商標)、ポリグリコール酸、ポリヒドロキシアルカノエート、コラーゲン、およびゼラチンから選択される一以上の物質を含む、請求項22に記載の組成物。
- 前記抗PD−1抗体が、抗原によって引き起こされた免疫細胞応答を調節する、請求項20に記載の組成物。
- 前記抗原が、前記抗PD−1抗体と同時に提示される、請求項24に記載の組成物。
- 前記抗原と抗PD−1抗体とが、固体支持体マトリクス上に固定されており、そして100μmまたは100μmより小さい間隔を空けて同時に提示される、請求項24に記載の組成物。
- Fab、F(ab’)2、Fv、scFvおよびdAbからなる群より選択される抗PD−1抗体フラグメントであって、
(a)配列番号26(CDRL1)、配列番号27(CDRL2)および配列番号28(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖;および
(b)配列番号23(CDRH1)、配列番号24(CDRH2)および配列番号25(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖
を含む、抗PD−1抗体フラグメント。 - Fab、F(ab’)2、Fv、scFvおよびdAbからなる群より選択される抗PD−1抗体フラグメントであって、
(a)配列番号32(CDRL1)、配列番号33(CDRL2)および配列番号34(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖;および
(b)配列番号29(CDRH1)、配列番号30(CDRH2)および配列番号31(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖
を含む、抗PD−1抗体フラグメント。 - Fab、F(ab’)2、Fv、scFvおよびdAbからなる群より選択される抗PD−1抗体フラグメントであって、
(a)配列番号38(CDRL1)、配列番号39(CDRL2)および配列番号40(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖;および
(b)配列番号35(CDRH1)、配列番号36(CDRH2)および配列番号37(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖
を含む、抗PD−1抗体フラグメント。 - Fab、F(ab’)2、Fv、scFvおよびdAbからなる群より選択される抗PD−1抗体フラグメントであって、
(a)配列番号53(CDRL1)、配列番号54(CDRL2)および配列番号55(CDRL3)に示される配列を有する相補性決定領域(CDR)L1、L2およびL3を含む軽鎖;および
(b)配列番号50(CDRH1)、配列番号51(CDRH2)および配列番号52(CDRH3)に示される配列を有する相補性決定領域(CDR)H1、H2およびH3を含む重鎖
を含む、抗PD−1抗体フラグメント。
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EP1576014B1 (en) | 2011-06-29 |
CA2508660C (en) | 2013-08-20 |
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