JP2020183435A - ワクチンとしての使用のためのガス入りの微小胞 - Google Patents
ワクチンとしての使用のためのガス入りの微小胞 Download PDFInfo
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- JP2020183435A JP2020183435A JP2020127322A JP2020127322A JP2020183435A JP 2020183435 A JP2020183435 A JP 2020183435A JP 2020127322 A JP2020127322 A JP 2020127322A JP 2020127322 A JP2020127322 A JP 2020127322A JP 2020183435 A JP2020183435 A JP 2020183435A
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- Prior art keywords
- acid
- formulation
- gas
- antigen
- dipalmitoyl
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Abstract
Description
共有結合した抗原(OVA)を含むガス入りの微小胞の調製
a)OVAのチオール化
OVA(6mg−133nmoles)を、PBE(リン酸バッファー25mM、150mM生理食塩水、1mM EDTA、pH8)に溶解させて、20mg/mLの溶液を得た。Traut試薬の溶液(2mg/mL−14.5mM)をPBE中に調製して、92μLのこの溶液(10equ.)をOVA溶液に添加した。生じた混合物を、撹拌しながら室温で1時間インキュベートした。この溶液を、PBE中で平衡化されたスピンカラム(Zebaスピンカラム2mL、Pierce,#89890)を通してスピンした。溶液の最終容量は約390μLであった。
ガス入りの微小胞の4つの製剤、すなわち、本発明に係る製剤A1およびA2および比較製剤B1およびB2を、以下の方法論を用いて調製した。それぞれの製剤における成分のタイプおよび量は、以下の表1に特定される。
DSPE−PEG−マレイミド(2.2mg−7.47μmoles)を、撹拌(ボルテックス)によって45℃でリン酸バッファー100mM pH6(0.2mL)に溶解させて、透明な溶液を得た。
DPPE−MPB(4.2mg−4.39μmoles)を、DSPC/パルミチン酸の混合物20mg(表1に示されるモル比)と混合して、撹拌(ボルテックス)によって60℃でエタノール中に溶解させて、透明な溶液を得た。溶媒を窒素下で蒸発させて、残渣を真空下で一晩乾燥させた。
ミューリンDCのインビトロ活性化:微小胞の製剤A1と比較B1との間の比較
脾臓由来マウスDC2.4細胞を、完全RPMI培地中で維持した。それらを、完全RPMI培地中で、ポリ−L−リジンによりコーティングされたミニトレイ培養プレートでインキュベートした。それから、5μlの微小胞製剤A1またはB1(1000微小胞:1DCの比を得るような希釈で再構成した)をウェルに添加して、培養プレートを転倒させて、37℃で24時間、上下逆にしてインキュベートした。培養上清中のTNF−αを、酵素結合免疫吸着測定法(ELISA)によって定量した。CD16/32に対するmAbを用いて細胞をブロッキングして、CD40に対するモノクローナル抗体を用いて染色して、GalliosフローサイトメーターおよびFlowJoソフトウェア(Tree Star Inc,Ashland,OR)を用いて解析した。
製剤A1およびA2とそれぞれの比較製剤B1およびB2との間の短期免疫応答の比較
100μlの異なる微小胞製剤(A1、A2、B1またはB2;100μlの懸濁液あたり4.5μgのOVAの濃度を与えるような生理食塩水の容量で再構成された、実施例1のバイアル中の前駆体)を用いて、尾の付け根に皮下でマウスを免疫化した。Limulus Amebocyte Lysateアッセイによってエンドトキシンレベルを測定して、OVAのμgあたり1エンドトキシン単位(EU)よりも低いことが分かった。2週間隔で3回の投与を行なって、最後の注入の14日後に脾臓を集めた。血清中のOVA特異的抗体の存在および力価を、以下のプロトコルに従ってELISAによって決定した。手短には、Maxisorbプレート(Nunc)を10μg/mL OVAでコーティングして、PBS−0.05% Tween 20−1% BSAでブロッキングして、滴定された投与量の血清の添加前に洗浄した。4℃での一晩のインキュベーションの後に、プレートを洗浄して、OVA特異的なIgGの存在を、HRPが結合した抗IgG抗体を用いたインキュベーションにより評価した。洗浄の後に、HRP基質を添加して、1M H2SO4を用いて数分後に反応を止めて、OD 450nmを測定した。得られた滴定曲線に基づいて(血清希釈(アゴニスト)vs.OD 450nm(応答))、シグモイドフィッティング曲線を作成した。それから、試験されたそれぞれの条件の比較を可能にする、それぞれの曲線に関して最大半量OD 450nmを与える血清希釈(力価)を計算した(EC50)。脾細胞を丸底96ウェルプレートに4・105細胞/ウェルで蒔いて、培地のみ、OVA(100μg/mL)、または、ポジティブコントロールとしてコンカナバリンA(1.5μg/mL)のいずれかとともにインキュベートして、インキュベートした。72時間のインキュベーションの後に細胞培養上清を集めて、酵素結合免疫吸着測定法(ELISA)によってIL−2、IFN−γ、IL−10、IL−4の存在について分析した。
T細胞応答の長期持続性の比較
100μlの微小胞製剤A2またはB1(100μlの懸濁液あたり4.5μgのOVAの濃度を与えるような生理食塩水の容量を用いて再構成された、実施例1のバイアル)を用いて、尾の付け根に皮下でマウスを免疫化した。2週間隔で3回の投与を行なって、最後の注入の14日後、2ヶ月後および6ヶ月後に血液および脾臓を集めた。脾臓の細胞懸濁液を、サイトカイン産生について、実施例3に説明したとおりに分析した。加えて、37℃で、PBS 0.1% BSA中でCFSEにより脾細胞をラベルして、それから、96ウェルプレート中に蒔く前に、冷却PBS 5%ウシ胎仔血清を用いて洗浄した。
OVA−Lm感染に対する、ワクチンにより誘導される長期の保護
100μlの微小胞製剤A2またはB1(100μlの懸濁液あたり4.5μgのOVAの濃度を与えるような生理食塩水の容量を用いて再構成された、実施例1のバイアル)を用いて、尾の付け根に皮下でマウスを免疫化した。2週間隔で3回の投与を行なった。最後の免疫化の6ヶ月後に、OVA(OVA−Lm)を安定的に発現する組み換えリステリア菌の50’000コロニー形成単位(CFU)のPBS溶液を静脈内注入した。脾臓および肝臓におけるOVA−Lm負荷を感染の4日後に分析した。PBS−0.1% NP40バッファー中の脾臓および肝臓を、70−μm細胞ストレーナーを用いてすり潰して、200μg/mlストレプトマイシンを含むBHIアガロースプレート上に段階希釈を蒔いて、37℃で24時間インキュベートした。細菌数を、脾臓または肝臓のCFU/mgとして表わす。脾臓の細胞懸濁液によって産生されたIFN−γを、実施例2に説明したとおりに定量化した。
Claims (17)
- 安定化膜を有するガス入りの微小胞の水性懸濁液を含む医薬製剤であって、
前記安定化膜は、(a)前記膜を形成する両親媒性成分に共有結合された抗原、(b)リン脂質、および、(c)脂肪酸を含み、
前記リン脂質と前記脂肪酸との間のモル比は、30/70よりも低い、
医薬製剤。 - 請求項1に記載の製剤であって、
前記モル比が、25/75またはそれよりも低い、
製剤。 - 請求項1に記載の製剤であって、
前記モル比が、20/80またはそれよりも低い、
製剤。 - 請求項1から3のいずれか一項に記載の製剤であって、
前記安定化膜におけるリン脂質の量は、前記膜を形成する成分の全体量に対して、50重量%未満である、
製剤。 - 請求項1から4のいずれか一項に記載の製剤であって、
前記抗原は、前記膜において、0.1%〜10%のモル量で存在する、
製剤。 - 請求項1から5のいずれか一項に記載の製剤であって、
前記微小胞内に含まれる前記ガスは、フッ素化ガスを含む、
製剤。 - 請求項10に記載の製剤であって、
前記ガスは、空気または窒素と混合される、
製剤。 - 請求項1から7のいずれか一項に記載の製剤であって、
前記リン脂質は、ジラウロイル−ホスファチジル−コリン(DLPC)、ジミリストイル−ホスファチジルコリン(DMPC)、ジパルミトイル−ホスファチジル−コリン(DPPC)、ジアラキドイル−ホスファチジルコリン(DAPC)、ジステアロイル−ホスファチジル−コリン(DSPC)、ジオレオイル−ホスファチジルコリン(DOPC)、1,2ジステアロイル−sn−グリセロ−3−エチルホスホコリン(エチル−DSPC)、ジペンタデカノイル−ホスファチジルコリン(DPDPC)、1−ミリストイル−2−パルミトイル−ホスファチジルコリン(MPPC)、1−パルミトイル−2−ミリストイル−ホスファチジルコリン(PMPC)、1−パルミトイル−2−ステアロイル−ホスファチジルコリン(PSPC)、1−ステアロイル−2−パルミトイル−ホスファチジルコリン(SPPC)、1−パルミトイル−2−オレイル−ホスファチジル−コリン(POPC)、1−オレイル−2−パルミトイル−ホスファチジルコリン(OPPC)、ジラウロイル−ホスファチジルグリセロール(DLPG)およびそのアルカリ金属塩類、ジアラキドイルホスファチジル−グリセロール(DAPG)およびそのアルカリ金属塩類、ジミリストイルホスファチジルグリセロール(DMPG)およびそのアルカリ金属塩類、ジパルミトイルホスファチジルグリセロール(DPPG)およびそのアルカリ金属塩類、ジステアロイルホスファチジルグリセロール(DSPG)およびそのアルカリ金属塩類、ジオレオイル−ホスファチジルグリセロール(DOPG)およびそのアルカリ金属塩類、ジラウロイルホスファチジン酸(DLPA)、ジミリストイルホスファチジン酸(DMPA)およびそのアルカリ金属塩類、ジパルミトイルホスファチジン酸(DPPA)およびそのアルカリ金属塩類、ジステアロイルホスファチジン酸(DSPA)、ジアラキドイルホスファチジン酸(DAPA)およびそのアルカリ金属塩類、ジラウロイル−ホスファチジルエタノールアミン(DLPE)、ジミリストイル−ホスファチジルエタノールアミン(DMPE)、ジパルミトイルホスファチジルエタノールアミン(DPPE)、ジステアロイルホスファチジル−エタノールアミン(DSPE)、ジオレイルホスファチジル−エタノールアミン(DOPE)、ジアラキドイルホスファチジル−エタノールアミン(DAPE)、ジリノレイルホスファチジルエタノールアミン、ジラウロイル−ホスファチジル−セリン(DLPS)、ジミリストイルホスファチジルセリン(DMPS)、ジアラキドイル−ホスファチジル−セリン(DAPS)、ジパルミトイルホスファチジルセリン(DPPS)、ジステアロイルホスファチジルセリン(DSPS)、ジオレオイルホスファチジルセリン(DOPS)、ジパルミトイルスフィンゴミエリン(DPSP)、ジステアロイルスフィンゴミエリン(DSSP)、ジラウロイル−ホスファチジルイノシトール(DLPI)、ジアラキドイルホスファチジルイノシトール(DAPI)、ジミリストイルホスファチジルイノシトール(DMPI)、ジパルミトイルホスファチジルイノシトール(DPPI)、ジステアロイルホスファチジルイノシトール(DSPI)、ジオレオイル−ホスファチジルイノシトール(DOPI)からなる群より選択される、
製剤。 - 請求項8に記載の製剤であって、
ペグ化リン脂質をさらに含む、
製剤。 - 請求項8または9に記載の製剤であって、
前記脂肪酸は、カプリン(n−デカン)酸、ラウリン(n−ドデカノン)酸、ミリスチン(n−テトラデカン)酸、パルミチン(n−ヘキサデカン)酸、ステアリン(n−オクタデカン)酸、アラキジン(n−エイコサン)酸、ベヘン(n−ドコサン)酸およびn−テトラコサン酸からなる群より選択される、
製剤。 - 請求項1から10のいずれか一項に記載の製剤であって、
前記抗原に共有結合した前記両親媒性成分は、リン脂質である、
製剤。 - 請求項1から11のいずれか一項に記載の製剤であって、
免疫調節治療での使用のための、
製剤。 - 請求項12に記載の使用のための製剤であって、
前記治療は、ワクチン接種を含む、
製剤。 - 請求項12に記載の使用のための製剤であって、
前記抗原は、ワクチン抗原である、
製剤。 - 請求項12から14のいずれか一項に記載の使用のための製剤であって、
前記治療は、寛容導入の治療を含む、
製剤。 - 請求項12から14のいずれか一項に記載の使用のための製剤であって、
前記治療は、免疫賦活の治療を含む、
製剤。 - 請求項1から16のいずれか一項に記載のガス入りの微小胞の懸濁液の前駆体であって、
生理的に許容できるガスの存在下で、生理的に許容できる水性担体中で再構成可能な、凍結乾燥された製剤の形態である、
前駆体。
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