JP2017169569A - Il−17a及びil−17fに結合する抗体分子 - Google Patents
Il−17a及びil−17fに結合する抗体分子 Download PDFInfo
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Abstract
【解決手段】ヒトIL−17A及びヒトIL−17Fに結合する中和抗体であって、重鎖を含み、重鎖の可変領域が、特定の配列を持つCDR−H1、CDR−H2、及びCDR−H3配列を含む中和抗体を提供する。更に、軽鎖の可変領域が、特定の配列を持つCDR−L1、CDR−L2、及びCDR−L3を含むヒトIL−17A及びヒトIL−17Fに結合する中和抗体。
【選択図】なし
Description
フェニルアラニン、チロシン及びトリプトファン(芳香族側鎖を有するアミノ酸)
リシン、アルギニン及びヒスチジン(塩基性側鎖を有するアミノ酸)
アスパラギン酸及びグルタミン酸(酸性側鎖を有するアミノ酸)
アスパラギン及びグルタミン(アミノ側鎖を有するアミノ酸)及び
システイン及びメチオニン(硫黄含有側鎖を有するアミノ酸)
を含む。同一性又は類似性の度合は、容易に計算できる(Computational Molecular Biology、Lesk、A.M.編、Oxford University Press、New York、1988;Biocomputing.Informatics and Genome Projects、Smith,D.W.編、Academic Press、New York、1993;Computer Analysis of Sequence Data、Part1、Griffin、A.M.and Griffin,H.G.編、Humana Press、New Jersey、1994;Sequence Analysis in Molecular Biology、von Heinje、G.、Academic Press、1987;及びSequence Analysis Primer、Gribskov、M.and Devereux,J.編、M Stockton Press、New York、1991)
大腸菌株INVαF’(Invitrogen)を、形質転換及び日常の培養成長に使用した。DNAの制限酵素及び修飾酵素は、Roche Diagnostics Ltd.及びNew England Biolabsから入手した。プラスミドの調整は、マキシプラスミド(Maxi Plasmid)精製キット(QIAGEN、カタログ番号12165)を使用して実施した。DNAシークエンシング反応は、ABI Prism Big Dye terminator sequencing kit(カタログ番号4304149)を使用して実施し、ABI 3100自動シークエンサー(Applied Biosystems)で実行した。データを、プログラムAutoAssembler(Applied Biosystems)を使用して分析した。オリゴヌクレオチドは、Invitrogenから入手した。IgGの濃度は、IgG会合ELISAを使用して決定した。
組換えIL−17A及びIL−17Fは、R&D Systemsから購入した。
組換えIL−17A/Fヘテロ二量体は、GSリンカーを使用して、IL−17A及びIL−17Fを連結することによって作製した。このヘテロ二量体は、以下の配列(配列番号19)を有した。
組換えカニクイザルIL−17F(配列番号20)
カニクイザルのL−17FをコードするDNA配列は、NdeI及びXhoIの制限部位に導入されたプライマーを使用して、PCRにより増幅した。PCR産物は、pCR4Blunt−TOPO内において連結し分解及びpET43.1aのNdeI/XhoI部位への連結前に、配列を確認した。
中和抗IL−17抗体の作製
メスのSprague Dawlyラットを組換えヒトIL−17(R&D systemsから購入)を用いて免疫化した。ラットは、100μlのフロイントのアジュバント中の20μgIL−17の免疫化を4回受けた。ヒトIL−17と結合した抗体225を、WO04/051268に記載の方法を使用して単離した。抗体225の重鎖可変領域(VH)及び軽鎖可変領域(VL)に関する遺伝子を単離し、逆転写PCRを介したクローニングの後でシークエンシングした。
抗体CA028_0496はIL−17及びIL−17F及びIL−17A/Fヘテロ二量体を中和する。
ヒーラ細胞
ヒーラ細胞におけるヒト組換えIL−17A及びヒト組換えIL−17Fに対する抗体CA028_0496の能力を試験し、抗体CDP435(WO06/054059)と比較した。ヒーラ細胞は、ATCC(ATCC CCL−2)の細胞バンクから得た。細胞を、10%ウシ胎児血清、ペニシリン、ゲンタマイシン及びグルタミンを添加したダルベッコ変法イーグル培地(DMEM)で成長させた。1×104の細胞を、96ウェルの平底組織培養プレートに播種した。細胞を一晩インキュベートし、アッセイバッファーで1回洗浄した。ヒトIL−17A(25ng/ml)又はヒトIL−17F(125ng/ml)のどちらかを、固定濃度のヒトTNFαの存在下でインキュベートし、この混合物を、抗体CA028_0496又は抗体CDP435とともにプレインキュベートした。その後、サイトカインと抗体とを、一晩インキュベートしたヒーラ細胞に加えた。細胞培養の上清におけるIL−6の産生は、細胞に加えたIL−17A/IL−17Fの量と比例していた。ヒトIL−6のレベルをELISAにより測定し、ヒトIL−6の公知の標準濃度と比較することによって定量化した。
ヒトミクログリア細胞(TCS Cellworks)を、平底の96ウェルプレートに、ウェル当たり5,000細胞で、全量100μlで播種し、プラスチックへの付着のため24時間放置した。この時、ヒト組換えIL−17A、ヒト組換えIL−17F、カニクイザル組換えIL−17F及びヒト組換えIL−17A/Fヘテロ二量体の滴定(5、1、0.2及び0.04μg/ml)を、10ng/mlのヒト組換えTNFαの存在下及び不在下で、ウェルに3重に加えた。対照ウェルは、刺激を含まず、IL−17A単独(100ng/ml)、TNFα単独並びにIL−17A及びTNFαを一緒に含む。すべてのサイトカインを、全容量が110μl/ウェルで加え、ウェルの全容量を210μlにした。抗体に関する実験において、細胞を、同じ方法で播種した。24時間後、抗体及びサイトカインを同時に加え、全最終容量200μlにおいて定められた最終濃度をもたらした。
抗体CA028_0496(ヒトIgG4定常領域)のIL−17A及びIL−17Fに対する親和性
BIA(Biamolecular Interaction Analysis)を、Biacore3000(Biacore AB)を使用して実施した。すべての実験は、25℃において実施した。Affinipure Fc Fragment goat anti−human IgG,Fc fragment specific(Jackson ImmunoResearch)を、アミン結合化学反応を介して、捕捉レベルおよそ6000反応単位(RU)まで、CM5 Sensor Chipに固定した。HBS−EPバッファー(10mM HEPES pH7.4、0.15M NaCl、3mM EDTA、0.005% Surfactant P20、Biacore AB)を、流速10μl/分で、ランニングバッファーとして使用した。固定抗ヒトIgG−Fcによって捕捉するために、抗体CA028_0496(1.81mg/ml)を10μl注入した。ヒトのIL−17A及びIL−17のイソ型を、50nMからサブnMに2重に希釈した捕捉CA028_0496により、流速30μL/分で滴定した。30μLの40mM HClを注入し、その後5μLの5mM NaOHを1度注入することによって、表面を再生した。
IL−17A、カニクイザルIL−17F及びIL−17A/Fヘテロ二量体に対する、抗体CA028_0496(マウスIgG1定常領域)の親和性
BIA(Biamolecular Interaction Analysis)を、Biacore3000(Biacore AB)を使用して実施した。
すべての実験は、25℃において実施した。Affinipure F(ab’)2 fragment goat anti−mouse IgG,Fc fragment specific(Jackson ImmunoResearch)を、アミン結合化学を介して、捕捉レベルおよそ6000反応単位(RU)まで、CM5 Sensor Chip(Biacore AB)に固定した。HBS−EPバッファー(10mM HEPES pH7.4、0.15M NaCl、3mM EDTA、0.005% Surfactant P20、Biacore AB)を、流速10μl/分で、ランニングバッファーとして使用した。固定抗マウスIgG、Fcによって捕捉するために、抗体CA028_0496(4ug/mlL)を10μl注入した。ヒトIL−17A、カニクイザルIL−17F及びヘテロ二量体A/Fを、25nMからサブnMに2重に希釈した、捕捉CA028_0496により、流速30μL/分で滴定した。流速10uL/分で10μLの40mM HClを注入し、その後5μLの5mM NaOHを1度注入することによって、表面を再生した。
また本発明の好ましい態様として以下の態様が含まれる。
(1)ヒトIL−17A及びヒトIL−17Fに結合する中和抗体であって、重鎖を含み、重鎖の可変領域が、CDR−H1に関して配列番号1で示される配列、CDR−H2に関して配列番号2で示される配列、及びCDR−H3に関して配列番号3で示される配列を含む、上記中和抗体。
(2)軽鎖を更に含み、軽鎖の可変領域が、CDR−L1に関して配列番号4で示される配列、CDR−L2に関して配列番号5で示される配列、及びCDR−L3に関して配列番号6で示される配列を含む、(1)に記載のヒトIL−17A及びヒトIL−17Fに結合する中和抗体。
(3)重鎖が配列番号9で示される配列又はそれと少なくとも90%の同一性を有する配列を含む、ヒトIL−17A及びヒトIL−17Fに結合する中和抗体。
(4)軽鎖を更に含み、軽鎖が配列番号7で示される配列又はそれと少なくとも90%の同一性を有する配列を含む、(3)に記載の中和抗体。
(5)配列番号9で示される配列を含む重鎖、及び配列番号7で示される配列を含む軽鎖を有する、(2)に記載の中和抗体。
(6)配列番号15で示される配列又はそれと少なくとも95%の同一性を有する配列を含む重鎖、及び配列番号11で示される配列又はそれと少なくとも95%の同一性を有する配列を含む軽鎖を有する、ヒトIL−17A及びヒトIL−17Fに結合する中和抗体。
(7)ヒトIL−17A及びヒトIL−17Fに結合し、ヒトIL−17A及びヒトIL−17F上にある、(5)に記載の抗体と同じエピトープに結合する中和抗体。
(8)ヒトIL−17A及びヒトIL−17Fに結合し、(5)に記載の抗体の、ヒトIL−17A及び/又はヒトIL−17F及び/又はIL−17A/Fヘテロ二量体への結合を交差ブロックし、並びに/或いは(5)に記載の抗体によって、ヒトIL−17A及び/又はヒトIL−17F及び/又はIL−17A/Fヘテロ二量体を結合することから交差ブロックされる、中和抗体。
(9)抗体が、抗体全体又はそれらの機能的に活性な断片又は誘導体である、(1)から(8)までのいずれかに記載の中和抗体。
(10)抗体断片が、ドメイン抗体、Fab、Fab’、F(ab’) 2 、scFv又はそれらのエピトープ結合断片である、(9)に記載の中和抗体。
(11)抗体又はその断片が、CDRグラフト抗体又は完全ヒト抗体である、(10)に記載の中和抗体。
(12)抗体又はその断片が、1種又は複数種のエフェクター分子に結合した、(1)から(11)までのいずれかに記載の抗体。
(13)(1)から(11)までのいずれかに記載の抗体の重鎖及び/又は軽鎖をコードする、単離DNA配列。
(14)(13)に記載の1種又は複数種のDNA配列を含む、クローニングベクター又は発現ベクター。
(15)ベクターが配列番号18で示される配列を含む、(14)に記載のベクター。
(16)ベクターが、配列番号14及び配列番号18で示される配列を含む、(15)に記載のベクター。
(17)(14)から(16)までのいずれかに記載の1種又は複数種のクローニングベクター又は発現ベクターを含む宿主細胞。
(18)(17)に記載の宿主細胞を培養するステップ、及び抗体を単離するステップを含む、(1)から(11)までのいずれかに記載の抗体の作製方法。
(19)(1)から(11)までのいずれかに記載の抗体と、1種又は複数種の医薬として許容可能な賦形剤、希釈剤又は担体とを組み合わせて含む、医薬組成物。
(20)他の活性成分をさらに含む、(19)に記載の医薬組成物。
(21)IL−17A及び/又はIL−17Fにより仲介される、或いはIL−17A及び/又はIL−17Fのレベルの上昇に伴う、病的障害の治療又は予防における使用のための、(1)から(11)までのいずれかに記載の抗体、或いは(19)又は(20)に記載の医薬組成物。
Claims (21)
- ヒトIL−17A及びヒトIL−17Fに結合する中和抗体であって、重鎖を含み、重鎖の可変領域が、CDR−H1に関して配列番号1で示される配列、CDR−H2に関して配列番号2で示される配列、及びCDR−H3に関して配列番号3で示される配列を含む、上記中和抗体。
- 軽鎖を更に含み、軽鎖の可変領域が、CDR−L1に関して配列番号4で示される配列、CDR−L2に関して配列番号5で示される配列、及びCDR−L3に関して配列番号6で示される配列を含む、請求項1に記載のヒトIL−17A及びヒトIL−17Fに結合する中和抗体。
- 重鎖が配列番号9で示される配列又はそれと少なくとも90%の同一性を有する配列を含む、ヒトIL−17A及びヒトIL−17Fに結合する中和抗体。
- 軽鎖を更に含み、軽鎖が配列番号7で示される配列又はそれと少なくとも90%の同一性を有する配列を含む、請求項3に記載の中和抗体。
- 配列番号9で示される配列を含む重鎖、及び配列番号7で示される配列を含む軽鎖を有する、請求項2に記載の中和抗体。
- 配列番号15で示される配列又はそれと少なくとも95%の同一性を有する配列を含む重鎖、及び配列番号11で示される配列又はそれと少なくとも95%の同一性を有する配列を含む軽鎖を有する、ヒトIL−17A及びヒトIL−17Fに結合する中和抗体。
- ヒトIL−17A及びヒトIL−17Fに結合し、ヒトIL−17A及びヒトIL−17F上にある、請求項5に記載の抗体と同じエピトープに結合する中和抗体。
- ヒトIL−17A及びヒトIL−17Fに結合し、請求項5に記載の抗体の、ヒトIL−17A及び/又はヒトIL−17F及び/又はIL−17A/Fヘテロ二量体への結合を交差ブロックし、並びに/或いは請求項5に記載の抗体によって、ヒトIL−17A及び/又はヒトIL−17F及び/又はIL−17A/Fヘテロ二量体を結合することから交差ブロックされる、中和抗体。
- 抗体が、抗体全体又はそれらの機能的に活性な断片又は誘導体である、請求項1から8までのいずれか一項に記載の中和抗体。
- 抗体断片が、ドメイン抗体、Fab、Fab’、F(ab’)2、scFv又はそれらのエピトープ結合断片である、請求項9に記載の中和抗体。
- 抗体又はその断片が、CDRグラフト抗体又は完全ヒト抗体である、請求項10に記載の中和抗体。
- 抗体又はその断片が、1種又は複数種のエフェクター分子に結合した、請求項1から11までのいずれか一項に記載の抗体。
- 請求項1から11までのいずれか一項に記載の抗体の重鎖及び/又は軽鎖をコードする、単離DNA配列。
- 請求項13に記載の1種又は複数種のDNA配列を含む、クローニングベクター又は発現ベクター。
- ベクターが配列番号18で示される配列を含む、請求項14に記載のベクター。
- ベクターが、配列番号14及び配列番号18で示される配列を含む、請求項15に記載のベクター。
- 請求項14から16までのいずれか一項に記載の1種又は複数種のクローニングベクター又は発現ベクターを含む宿主細胞。
- 請求項17に記載の宿主細胞を培養するステップ、及び抗体を単離するステップを含む、請求項1から11までのいずれか一項に記載の抗体の作製方法。
- 請求項1から11までのいずれか一項に記載の抗体と、1種又は複数種の医薬として許容可能な賦形剤、希釈剤又は担体とを組み合わせて含む、医薬組成物。
- 他の活性成分をさらに含む、請求項19に記載の医薬組成物。
- IL−17A及び/又はIL−17Fにより仲介される、或いはIL−17A及び/又はIL−17Fのレベルの上昇に伴う、病的障害の治療又は予防における使用のための、請求項1から11までのいずれか一項に記載の抗体、或いは請求項19又は請求項20に記載の医薬組成物。
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