JP6853317B2 - 高度に低下された受容体結合親和性を有するサイトカインを含むフソカイン - Google Patents
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Description
XCL1は、CD8+T細胞、Th1細胞分極化CD4+T細胞及びNK細胞により分泌される93アミノ酸のケモカインである。それは、専ら樹状細胞により発現されるケモカイン受容体であるXCR1と相互作用する。マウスにおいてXCR1は、脾臓CD11c+CD8α+樹状細胞の多数で発現されるが、CD8α−樹状細胞では非常にわずかな一部だけが、この受容体を発現する(Dorner et al. 2009)。XCR1は、マウスCD8α+樹状細胞に相同的な哺乳動物細胞(ヒト細胞を含む)の保存された選択マーカである(Crozat et al. 2010)。興味深いこととして、この樹状細胞サブセットに対するI型インターフェロン(IFNα/β)の作用が、マウスの体内で成長する腫瘍の生来の免疫認識に極めて重要であることが示されている(Fuertes et al. 2011)。
肝臓及び活性化調節ケモカイン(LARC)、マクロファージ炎症性タンパク質−3α(MIP−3α)またはエクソダス−1としても知られるCCケモカインCCL20は、主に肝臓及びリンパ球様組織において発現される96アミノ酸のタンパク質である(Hieshima et al., 1997)。分泌によりCCL20は、Gタンパク質共役受容体(GPCR)1ファミリーに属するCCケモカイン受容体6(CCR6)への結合により活性を発揮する(Baba et al., 1997)。CCR6発現は、異なる白血球サブセット上で報告されているが、Th17細胞集団について最もよく文書化されている(Singh et al., 2008)。正常なTh17機能は、結核菌(Khader et al., 2007)、クレブシエラ・ニューモニエ(Ye et al., 2001)及び百日咳菌(Higgins et al., 2006)をはじめとする様々な病原に対する防御的免疫性に絶対必要である。
TNFαは、細胞毒性、免疫細胞の調節及び炎症反応の仲介といった広範囲の生物活性を有するサイトカインである。それは、223のアミノ酸からなる自己組織化する非共有結合性ホモトリマーのII型膜貫通タンパク質である。TNFαは、膜結合性で、ならびにTNFα変換酵素(TACE。ADAM17とも称される)による76のアミノ末端アミノ酸(プレシークエンス)のタンパク質分解性切断後に細胞膜から放出される可溶性のタンパク質として活性である。それは、2種の別個の受容体、すなわちTNF−R1(p55)及びTNF−R2(p75)を通してシグナル伝達するが、それらは両者ともリガンド結合細胞外ドメイン内にシステインリッチモチーフを有する膜貫通糖タンパク質である。その細胞外の相同性にもかかわらず、それらは、別個の細胞内ドメインを有し、それゆえ異なるTNF活性をシグナル伝達する(Hehlgans & Pfeffer, 2005)。本発明者らは、以前にBoschert et al., 2010により記載された通り、GGGGSリンカーを介して連結された3つのTNFモノマーからなる一本鎖バリアント(scTNF)を作製した。
フソカインのクローニング及び生成
XCL1/IFNα2−Q124R融合タンパク質のクローニング
XCL1オープンリーティングフレームを、PCRにより、エクスパンド・ハイ・フィデリティー・PCRシステム(Expand High Fidelity PCR system)(ロッシュ・ダイアグノスティックス)及び以下のプライマーを用いて、XCL1コード化プラスミドMR200473(Origen Inc.)から合成した:
順方向: 5’GGGGGGGAATTCATGAGACTTCTCCTCCTGAC3’
逆方向:5’GGGGGGTCCGGAGGCCCAGTCAGGGTTATCGCTG3’
PCR産物をEcoRI及びBspEIにより消化して、pMET7 SIgK−HA−1R59B−His−PAS−ybbr−IFNA2−Q124Rベクター内のナノボディーをコードするEcoRI−BspEIフラグメントに置換した(PCT/EP2013/050787)。
Hek293T細胞を、標準的なリポフェクタミン法(インビトロジェン)を利用して、タンパク質融合構築物でトランスフェクトした。トランスフェクションの48時間後、培地を採取して、−20℃で貯蔵した。精製ナノボディーGFP−IFNα2−Q124R調製物(PCT/EP2013/050787に記載)を標準として用いて、IFN活性を、記載された通り(Uze et al. J. Mol. Biol. 1994)ヒトHL116及びネズミLL171細胞株でアッセイした。
成熟したヒトIL−1β/CCL20融合タンパク質をコードするコドン最適化配列を、遺伝子合成(インビトロジェン・ジーン・アート(Invitrogen Gene Art))により作製した。手短に言えば、SigKリーダーペプチドを先頭としN末端HAを備えた成熟ヒトIL−1βタンパク質配列を、C末端で13×GGSリンカー配列に、そしてその後、C末端HISタグを有する成熟ヒトCCL20の配列に融合した(図3)。
IL−1β−CCL20融合タンパク質を、HEK293T細胞内で産生した。小規模生成のために、HEK293T細胞を、10%FCSを補充されたDMEM中の400,000細胞/ウェルとして6ウェルプレートに播種した。24時間後に、培地を低濃度血清の培地(DMEM/5%FCS)と交換し、直鎖PEIを用いて細胞をトランスフェクトした。手短に言えば、DMEM 160μl中で発現ベクター1μgをPEI 5μgと混和し、室温で10分間インキュベートし、ウェルに滴下することにより、PEIトランスフェクションミックスを調製した。24時間後に、トランスフェクトされた細胞をDMEMで洗浄して、タンパク質生成のためにオプティメム/ウェル 1.5mlで層を形成させた。馴化培地を48時間後に回収し、0.45μフィルターでろ過して、−20℃で貯蔵した。馴化培地中のIL−1β量を、製造業者の使用説明書(R&Dシステムズ)に従ってELISAにより測定した。
野生型(WT)、L86S及びL86Nレプチンのコード配列を、PCRにより、以下のプライマーを用いて、それぞれWTレプチン、レプチンL86SまたはレプチンL86Nを発現するpMet7プラスミドから合成した:
順方向 5’−GCAGATCTGTCGACATCCAGAAAGTCCAGGATGACACC−3’,
逆方向 5’−CGATGCGGCCGCACATTCAGGGCTAACATCCAACTGT−3’。
これにより、レプチンコード配列の、それぞれアミノ末端及びカルボキシ末端にBgIII及びNotI部位を導入する。PCR産物をBgIII及びNotIで消化して、BgIII及びNotIにより開かれたpMET7−SIgK−HA−scTNF WT−6×GGS−FLAG(野生scTNFは、遺伝子合成により作製された。ジーンアート)にクローニングして、6×GGSとFLAGの間に存在させた。これにより、pMET7−SIgK−HA−scTNF WT−6×GGS−mレプチン−FLAG、pMET7−SIgK−HA−scTNF WT−6×GGS−mレプチンL86S−FLAG及びpMET7−SIgK−HA−scTNF WT−6×GGS−mレプチンL86N−FLAGを作製した。
標準的なリン酸カルシウム沈殿法を利用して、HekT細胞を異なる融合タンパク質構築物でトランスフェクトした。トランスフェクションの48時間後、培地を採取して、−20℃で貯蔵した。濃度を、市販のhTNFα ELISA(DY210、R&Dシステムズ)で測定した。
Hek293T、HL116及びLL171細胞株を、10%FCSを補充したDMEMで生育させた。Ba/F3−mLR及びBa/F3−mLR−TNFR1ΔCyt細胞を、10%熱活性化FCS及び100ng/mlレプチンを補充されたRPMIに保持した。
ホスホSTAT1アッセイ
単一細胞の懸濁液を、C57BI/6マウスから単離された脾臓から調製した。赤血球を、赤血球細胞溶解緩衝液(ロンザ)を用いて除去した。脾臓細胞を、37℃のRPMI5%ウシ胎仔血清中、マウスIFNα/βまたはXCL1−IFNα2−Q124R融合タンパク質で30分間処理し、その後、BDバイオサイエンシズ の使用説明書に従って、アレクサフロール488(Alexa Fluor 488)標識抗マウスCD11c(eバイオサイエンス #53−0114−80)とAPC標識抗マウスCD8α(BDバイオサイエンス #553035)、または抗マウスCD11cとアレクサ488標識抗マウスCD8αと共にBDホスフローPE(BD phosflow PE)マウス抗STAT1(pY701)で標識した。BDファクスカントを用いてFACSデータを得て、いずれかのディーバ(Diva)(BDバイオサイエンシズ)ソフトウエアを用いて解析した。
IL−1R活性化を評価するために、本発明者らは、IL−1Rを安定発現するHEK−ブルー(HEK−Blue)(商標)IL−1β細胞(インビボゲン)を用い、それらをNF−κBルシフェラーゼレポータージーンで一過性にトランスフェクトした。手短に言えば、HEK−ブルー(商標)IL−1β細胞を、96ウェルプレート中の培地(DMEM/10%FCS)に播種し(10000細胞/ウェル)、翌日、リン酸カルシウム沈殿法を利用し、示された量の発現プラスミド及び5ng/ウェル 3κB−Lucレポータージーンプラスミドでトランスフェクトした(Vanden Berghe et al., 1998)。トランスフェクションの24時間後、培地を飢餓培地(DMEM)に交換し、トランスフェクションの48時間後、細胞にIL1−CCL20融合タンパク質を6時間導入した。導入後に、細胞を溶解して、溶解物中のルシフェラーゼ活性を、ベルトールド・セントロLB960(Berthold centro LB960)ルミノメータによるプロメガ・ホタルルシフェラーゼアッセイシステムを用いて測定した。
Ba/F3−mLR細胞株を、pMet7−mLRベクターでのBa/F3細胞の電気穿孔により作製した。安定発現する細胞を、IL−3の代わりにレプチンで生育することにより選択した。実際にBa/F3細胞の生育は、IL−3に依存するが、それらの細胞がmLRを発現する場合には、それらはレプチンでも増殖する。Ba/F3−mLR−TNFR1ΔCyt細胞株を得るために、Ba/F3−mLR細胞をpMet7−HA−hTNFR1ΔCyt及びpIRESプロ2(pIRESpuro2)(クロンテック)で共トランスフェクトし、その後、hTNFR1ΔCyt発現細胞のプロマイシン選択及びFACS選別を行った。
マウス脾臓細胞を、1nM XCL1−IFNα2−Q124Rまたは10,000単位/mlマウスIFNα/βで30分間処理した。その後、細胞を固定して透過処理し、抗ホスホSTAT1(PE)、抗CD11c(アレクサフロール488)及び抗CD8α(APC)で染色して、FACSにより解析した。図2は、マウスIFNα/βが解析された全ての脾臓細胞サブセットにおいてSTAT1リン酸化を誘導したことを示している。反対にXCL1−IFNα2−Q124R融合タンパク質は、CD11c+CD8α+サブセットに属する細胞の多数、及びCD11c+CD8α−サブセットに属する細胞の少数だけにIFNの応答を誘導した。XCL1−IFNα2−Q124R融合タンパク質に応答する脾臓細胞サブセットの分布は、XCL1受容体であるXCR1の予測された分布と完全に一致する(Dorner et al. 2009)。
IL−1Rを安定発現するHEK−ブルー(商標)IL−1β細胞を、NF−κBレポータージーンプラスミド(5ng/ウェル)及び空ベクターまたはhCCR6発現プラスミド(10ng/ウェル)で一過性にトランスフェクトした。次に擬トランスフェクト及びCCR6トランスフェクトされた細胞を、野生型または変異型IL1β−CCL20融合タンパク質(25ng/ml)で6時間処理し、その後、細胞を溶解して、NF−κBレポータージーン活性を測定した。図4Aから明白な通り、CCR6を発現する細胞は、擬トランスフェクト細胞に比較して、全ての検討された変異型IL1β−CCL20融合タンパク質に対してNF−κBレポータージーン活性上昇として応答した。標的効果が最も明白であったIL−1β−Q148G変異体の効果をより詳細に評価するために、擬ランスフェクトされた、またはCCR6を発現するHEK−ブルー(商標)IL−1β細胞を、漸増量の野生IL−1βまたはIL−1βQ148G−CCL20融合タンパク質で6時間処理した。図4Bは、CCR6の過剰発現が野生IL−1β−CCL20融合物の活性を上昇させたが、IL−1βQ148G−CCL20融合物に関してはより強い増強効果を有したことを実証している。標的効果は、IL1−β−CCL20が12.5ng/mlで細胞に適用された場合に最も顕著であった(図4C)。
示された量のレプチンまたはレプチン−scTNF融合タンパク質での刺激の4日後に、Ba/F3−mLR及びBa/F3−mLR−TNFR1ΔCyt細胞の増殖を評価した。図6Aに示される通り、細胞株は両者とも、熱非活性化血清のみを補充された生育培地では増殖しない。その上、レプチンによるBa/F3増殖を誘導する能力は、レプチンがscTNFに結合された場合に低下する。野生レプチン内のL86をセリン(L86S)またはアスパラギン(L86N)のいずれかの中へ突然変異させると、マウスレプチン受容体への親和性が、それぞれ中等度または強度に低下される。この親和性低下は、それぞれレプチンL86S対L86Nで、Ba/F3−mLR細胞増殖誘導の3倍対10倍弱い誘導になる。細胞内ドメイン(hTNFR1ΔCyt)を欠くヒトTNF−R1によるBa/F3−mLR細胞のさらなるトランスフェクションは、膜結合細胞外マーカとして機能し得る非機能的受容体を誘導する。明らかに、scTNFに結合されたL86S及びL86Nレプチン変異体での刺激による増殖応答が、hTNFR1ΔCytを発現するBa/F3−mLR細胞において完全に回復される(図6B)。
Bachemら(Frontiers in Immunology 3, 1−12. 2012)によれば、XCR1発現細胞は、CD11c+CD8α+脾臓細胞集団の多数及びCD11c+CD8α−脾臓細胞集団の少数部分を示す。C57BI/6マウスに、示された量のXCL1−IFNα2−Q124Rまたは1,000,000単位の天然ネズミIFNα/βまたはPBSを静脈注射した。45分後に、脾臓細胞をFACSにより、以下の細胞集団中のP−STAT1について解析した:CD11c−CD8α−、CD11c−CD8α+、CD11c+CD8α+、CD11c+CD8α−。結果を図7に示す。これらの結果から、融合構築物が細胞集団の微量分画(細胞全体の約0.1%)を標的とし、それらにおいて応答を誘導し得るが、マーカを発現しないIFN感受性細胞が影響を受けないことが明らかである。事実、野生型INFもまた、CD11c+CD8α−細胞に影響を及ぼしているが、それらの細胞は、融合構築物による影響を受けておらず、この融合体の特異的作用が明瞭に立証される。
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Claims (6)
- 少なくとも2種のサイトカインを含む融合タンパク質を含む組成物であって、前記サイトカインが、CCL20及びIL−1βであり、前記IL−1βが、野生型IL−1βと比較してその受容体に対する結合活性を低下させる変異を含み、かつ前記CCL20が、細胞特異的な標的化を提供し、かつ標的細胞上の変異したIL−1βの活性を回復する野生型サイトカインである、組成物。
- GGSリンカーをさらに含む、請求項1に記載の組成物。
- 前記IL−1βの変異が、R120G、Q131G、H146A、Q148G、およびK209Aから選択される、請求項1に記載の組成物。
- 少なくとも2種のサイトカインを含む融合タンパク質を含む細胞における免疫応答を刺激するための組成物であって、前記サイトカインは、CCL20及びIL−1βであり、前記IL−1βが、野生型IL−1βと比較してその受容体に対する結合活性を低下させる変異を含み、かつ前記CCL20が、細胞特異的な標的化を提供し、かつ標的細胞上の変異したIL−1βの活性を回復する野生型サイトカインである、組成物。
- GGSリンカーをさらに含む、請求項4に記載の組成物。
- 前記IL−1βの変異が、R120G、Q131G、H146A、Q148G、およびK209Aから選択される、請求項4に記載の組成物。
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