JP6670297B2 - 抗セラミド抗体 - Google Patents
抗セラミド抗体 Download PDFInfo
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- JP6670297B2 JP6670297B2 JP2017506827A JP2017506827A JP6670297B2 JP 6670297 B2 JP6670297 B2 JP 6670297B2 JP 2017506827 A JP2017506827 A JP 2017506827A JP 2017506827 A JP2017506827 A JP 2017506827A JP 6670297 B2 JP6670297 B2 JP 6670297B2
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- ceramide
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Description
本出願は、2014年8月7日に出願された米国仮特許出願第62/034,453号の優先権を主張する。
スフィンゴ脂質、具体的にはスフィンゴミエリンと強く会合するコレステロールから構成され、無秩序液体バルク細胞膜内で秩序液体ドメインを形成している特徴的な細胞膜マイクロドメインである液状ラフト。ラフトは、タンパク質および脂質組成が周囲の膜と異なり、多数のグリコシルホスファチジルイノシトール(GPI)アンカー蛋白質、Srcファミリーの二重アシル化チロシンキナーゼ、および膜貫通タンパク質を含むシグナリング分子を収容する。さらにラフトは、抗原と遭遇した際に、B細胞受容体(BCR)を含む複数の受容体が活性化するとき、その中またはその外へ移動する部位としての役割を果たす。これらの移動事象がマルチシグナル伝導カスケードにとって重要であることが、エビデンスより示唆される。
最近、リーベルキューン陰窩の底部より1〜3位に位置する細胞周期の陰窩底部円柱(CBC)細胞が腸幹細胞集団として定義されている。この細胞群は絶え間なく増殖および分化し、絨毛先端からの腸細胞およびその他の分化上皮細胞の生理的な損失を補充することにより、粘膜の解剖学的および機能的完全性を維持する。陰窩−絨毛単位の恒久的破壊にはこのコンパートメントの完全な、またはほぼ完全な枯渇が必要であるとみられる一方で、生存幹細胞クロノーゲンは、1陰窩あたり1個であっても、完全に機能的な陰窩を再生することが可能である。
本願のさらなる他の態様は、それを必要とする対象における細胞死を阻害する方法であって、本願の抗セラミド抗体またはその抗原結合性フラグメントの治療的に有効な量を対象に投与することを含む方法に向けられる。一部の実施形態においては、抗セラミド抗体は抗セラミドscFvである。
抗体、またはその抗原結合性フラグメントは、ボーラスとして、または一定の時間をかけた連続注入による静脈内投与、筋肉内、腹腔内、脳脊椎液内(intracerobrospinal)、皮下、動脈内、滑液包内、くも膜下腔内、経口、局所または吸入経路などの既知の方法で対象に投与してもよい。
(ハイブリドーマからの2A2の可変L鎖およびH鎖のクローニング)
2A2ハイブリドーマ細胞を遠心分離により回収し、RNA精製キットを用いて総RNAを抽出した。この総RNAをcDNA合成に用い、「ファージディスプレイマニュアル」で公表されたプライマーセットを用いて2A2のV領域を最終的に分離した。図1Aは、2A2の可変H鎖(VH)およびL鎖(VL)配列を示す。
通常、げっ歯類抗体はヒトに対して免疫原性となり、かつHAMA(ヒト抗マウス抗体)応答またはアナフィラキシーショックを含む非常に重篤な副作用を引き起こす可能性がある。この問題を克服するため、抗体操作を用いて非ヒト抗体をヒト化している。したがって、CDR移植法を用いて2A2のVLおよびVHをヒト化した。
本来2A2 mAbはマウスIgMである。IgG1は血清中で最も豊富であり(9mg/mL)、その半減期(21日)は他の抗体のいずれもよりも長く、かつ現在最も流通している治療抗体はIgG1フォーマットであるので、IgM抗体はIgG1フォーマットに変換される。哺乳類発現ベクターでヒト化2A2 IgG1を構築するために、pOptiVECおよびpcDNA3.3(インビトロジェン)ベクターを用いた。
典型的なベクターは、幅広い哺乳類細胞における組換えタンパク質の高レベル発現を目的として、ヒトサイトメガロウイルス(CMV)最初期プロモーター/エンハンサーを含有する。ヒト化2A2 IgG1を構築するために、マウス2A2の3つのCDRをそれぞれ有するヒト可変L鎖およびH鎖を合成し、これら2つのDNAフラグメントを、PCRによりヒト定常L鎖およびH鎖に結合した。最後に、ヒト化2A2 L鎖をpcDNA3.3 TOPOにクローニングし、またヒト化2A2 H鎖をpOptiVEC TOPO抗体発現ベクターにクローニングした。ヒト2A2 IgG1の配列を図2A〜Bに示すが、ヒト定常L鎖の第1アミノ酸(Arginine、赤い網掛け)は、全ヒト化L鎖の構築の際に欠失したことが示された。これらのヒト2A2 Ab発現ベクターを構築した後、DNAプラスミドを、CHO由来DHFR陰性DG44細胞にコトランスフェクトして、2A2 hIgG1抗体を産生する安定した細胞株を作製した。
高レベルの抗体を産生する細胞株を得るため、CD OptiCHO培地およびジェネティシン500μg/mLを含有するCD OptiCHO培地を用いて2回の選択を行い、続いて、96ウェルプレート内の半固形培地でMTXゲノムの増幅選択および2回の単一細胞クローンの選択を行い、安定にトランスフェクトされた細胞のプールを選択した。ELISA測定定量により抗体の発現レベルをスクリーニングし、選択したh2A2IgG1−CHO細胞(G3A10、C5G6およびD5F11)株を徐々にスケールアップした。
マウスにおける反復投与試験を目的としたモノクローナル抗体パネルを生成し、免疫原性を検討した。抗原(アルブミン結合ω−COOH C16−セラミド)を用いて、抗セラミドMabの選択を目的としたELISAによるハイブリドーマのスクリーニングを実施した。BSAおよびアルブミン結合ω−COOH C16ジヒドロセラミドに対して、陽性ヒットをカウンタースクリーニングした。次に、Mabの生物学的試験(Jurkat細胞アポトーシスのインビトロ阻害、放射線GI症候群のインビボ阻害)を実施した。9H10、9H11、9A2、7B10、6B5および6C8と命名したクローンを試験用に選択したところ、9A2を除く全てがインビトロで生物学的活性を示した。表1に示すように、クローンのパネルはC16:0カルボキシセラミド−BSAと優先的に結合した。Ag1はC16:0カルボキシセラミド−BSA300ng/ウェルでコーティングし、Ag2はC−16:0ジヒドロカルボキシセラミド−BSA300ng/ウェルでコーティングし、Ag3は非結合BSA(シグマA6003)300ng/ウェルでコーティングした。
Mabの有効性データに基づき、6B5のCDRを(2A2と共に)選択し、操作して単鎖Fvとした。2つの単鎖(sc)Fv構成体を操作してscFvを発現させ、有効性試験用の精製scFvを提供した。6B5 scFvは容易に発現および精製された。Mabと同様、Jurkat細胞アポトーシスのインビトロ阻害および放射線GI症候群のインビボ阻害を用いて、scFvの生物学的試験を実施した。図5は、マイクロコロニー測定により、6B5 IgGが陰窩死より保護することを示す。図7は、scFvがJurkat細胞アポトーシスを阻害することを示す。図8は、6B5 scFvを15Gy被曝より15分前に投与した場合、インビボGI陰窩枯渇から保護することを示すことの例示である。図9は、6B5 scFvを15Gy被曝より24時間後に投与した場合、インビボGI陰窩枯渇から保護することを示す。
C57BL/6マウス(MHC H2bハプロタイプ)に対し、生理食塩水、ヒト化抗セラミドh2A2 50mg/kgまたは抗セラミドscFv 6B5 7.5mg/kgを、指示された投与経路および投与スケジュールにより投与した。投与は1100cGy分割線量全身照射(TBI)の15分前に開始した。次にマウスは、TBIより16〜20時間後に、B10.BRドナーマウス(MHC H2k2ハプロタイプ)に由来する5×106個の同種骨髄(BM)またはBMおよび2×106個の同種CD5+ナイーブT細胞からなる同種骨髄移植を受けた。マウスの生存を連日モニタリングした。データは、90日生存を表すと判定された10日目の生存を表す。投与予定日0、4および8日目に生理食塩水を静脈内投与したところ、10日後の生存率は30%となった。投与予定日0、4および8日目にh2A2モノクローナル抗体を静脈内投与したところ、10日後の生存率は100%となった(生理食塩水対照に対してp<0.001)。投与予定日0、4および8日目にscFVを静脈内投与したところ、10日後の生存率は60%となった(生理食塩水対照に対してp<0.05)。投与予定日0、2、4、6および8日目に生理食塩水を皮下投与したところ、10日後の生存率は0%となった。投与予定日0、2、4、6および8日目に生理食塩水を皮下投与したところ、10日後の生存率は100%となった(生理食塩水対照に対してp<0.001)。
Claims (17)
- H鎖可変領域(VH)及びL鎖可変領域(VL)を含む、抗セラミド抗体またはその抗原結合性フラグメントであって:
前記VHは、配列:GYTFTDHTIH(配列番号1)を含むH鎖可変領域CDR1、配列:YNYPRDGSTKYNEKFKG(配列番号2)を含む前記H鎖可変領域CDR2、及び、配列:GFITTVVPSAY(配列番号3)を含むH鎖可変領域CDR3を含み、並びに、
前記VLは、配列:RASKSISKYLA(配列番号4)を含むL鎖可変領域CDR1、配列:SGSTLQS(配列番号5)を含むL鎖可変領域CDR2、及び、配列:QQHNEYPWT(配列番号6)を含むL鎖可変領域CDR3を含む、
前記抗体またはその抗原結合性フラグメント。 - 請求項1に記載の抗セラミド抗体またはその抗原結合性フラグメントであって:
配列番号7を含むH鎖可変領域配列と少なくとも約90%の配列同一性を有する配列;および、
配列番号8を含むL鎖可変領域配列と少なくとも約90%の配列同一性を有する配列、
を含む前記抗体またはその抗原結合性フラグメント。 - 抗セラミド抗体またはその抗原結合性フラグメントであって:
配列:QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYNYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCAKGFITTVVPSAYWGQGTLVTVSA(配列番号48)であるH鎖可変領域配列;および、
配列:DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK(配列番号8)であるL鎖可変領域配列、
を含む前記抗体またはその抗原結合性フラグメント。 - 請求項1〜3のいずれか一項に記載の抗セラミド抗体またはその抗原結合性フラグメントであって、前記抗体がモノクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、およびscFvからなる群から選択される、前記抗体またはその抗原結合性フラグメント。
- 請求項4に記載の抗セラミド抗体であって、scFvである前記抗体。
- 請求項1に記載の前記抗セラミド抗体またはその抗原結合性フラグメントと同じ抗原決定基と結合する、抗セラミド抗体またはその抗原結合性フラグメント。
- 請求項1〜6のいずれか一項に記載の抗セラミド抗体またはその抗原結合性フラグメントであって、抗体またはその抗原結合性フラグメントは、吸入、静脈内、筋肉内、腹腔内、脳脊椎液内、皮下、滑液包内、くも膜下腔内、経口、または局所投与のために調剤されている、前記抗体またはその抗原結合性フラグメント。
- それを必要とする対象においてアポトーシスを阻害するための薬剤の製造における、請求項1〜7のいずれか一項に記載の抗セラミド抗体またはその抗原結合性フラグメントの使用。
- 請求項8に記載の使用であって、前記アポトーシスが移植片対宿主病、放射能疾患、GI症候群および自己免疫性疾患からなる群から選択される疾患に随伴する前記使用。
- 請求項9に記載の使用であって、前記疾患が放射能疾患またはGI症候群であり、かつ前記抗セラミド抗体またはその抗原結合性フラグメントが、前記対象が放射線に被曝する前に投与される、前記使用。
- 請求項9に記載の使用であって、前記疾患が移植片対宿主病であり、かつ前記対象が移植を受ける前に前記抗セラミド抗体またはその抗原結合性フラグメントが投与される前記使用。
- 請求項11に記載の使用であって、前記移植片が骨髄移植片である前記使用。
- 対象の透過線への被曝後のGI症候群である該対象におけるアポトーシスの緩和のための薬剤の製造における、請求項1〜7のいずれか一項に記載の抗セラミド抗体またはその抗原結合性フラグメントの使用。
- 請求項13に記載の使用であって、前記対象の透過線への被曝直後に前記抗セラミド抗体またはその抗原結合性フラグメントを投与する前記使用。
- 請求項13に記載の使用であって、前記対象の透過線への被曝後24時間以内に前記抗セラミド抗体またはその抗原結合性フラグメントを投与する前記使用。
- 対象が移植を受ける前または前記対象が移植を受けた後のGvHD発症前の、該対象におけるGvHDにおけるアポトーシスの阻害のための薬剤の製造における、請求項1〜7のいずれか一項に記載の抗セラミド抗体またはその抗原結合性フラグメントの使用。
- 請求項16に記載の使用であって、前記移植片が骨髄移植片である前記使用。
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