JP6453063B2 - アジュバント組成物 - Google Patents
アジュバント組成物 Download PDFInfo
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- JP6453063B2 JP6453063B2 JP2014244421A JP2014244421A JP6453063B2 JP 6453063 B2 JP6453063 B2 JP 6453063B2 JP 2014244421 A JP2014244421 A JP 2014244421A JP 2014244421 A JP2014244421 A JP 2014244421A JP 6453063 B2 JP6453063 B2 JP 6453063B2
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Classifications
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Description
[1]配列番号3に示される塩基配列からなる一本鎖核酸Aと、配列番号4に示される一本鎖核酸Bにより形成されている二本鎖核酸を含有することを特徴とするアジュバント組成物。
[2]一本鎖核酸Aおよび一本鎖核酸Bが化学合成されていることを特徴とする前記[1]に記載のアジュバント組成物。
[3]一本鎖核酸Aおよび一本鎖核酸Bが、化学合成された複数のフラグメントをライゲーションにより連結させて作製されていることを特徴とする前記[1]または[2]に記載のアジュバント組成物。
[4]一本鎖核酸Aおよび一本鎖核酸Bのいずれの末端にもリン酸基が結合していないことを特徴とする前記[1]〜[3]のいずれかに記載のアジュバント組成物。
[5]一本鎖DNAを構成するヌクレオチドの全部がホスホロチオエート修飾されていることを特徴とする前記[1]〜[4]のいずれかに記載のアジュバント組成物。
[6]前記[1]〜[5]のいずれかに記載のアジュバント組成物および抗原または抗原性成分を含むワクチン組成物。
[7]前記抗原ががん抗原である前記[6]に記載のワクチン組成物。
本発明は、配列番号1に示される塩基配列およびその相補配列からなる二本鎖RNAと、配列番号2に示される塩基配列からなる一本鎖DNAとが結合している核酸を含有するアジュバント組成物を提供する。本発明のアジュバント組成物に含有される核酸(以下、「本発明の核酸」と記す)は、配列番号1に示される塩基配列およびその相補配列からなる二本鎖RNAと、配列番号2に示される塩基配列からなる一本鎖DNAとが結合している核酸であればよい。本発明の核酸の二本鎖RNA部分はTLR3活性化能を有しており、一本鎖DNA部分は樹状細胞のエンドソームに送達されることが確認されている。したがって、本発明の核酸は、樹状細胞のエンドソームに効率よく送達され、TLR3を活性化することにより免疫機能を賦活化することができる。
本発明は、上記本発明のアジュバント組成物と抗原または抗原性成分を含むワクチン組成物を提供する。抗原または抗原性成分としては、ウイルス抗原、細菌抗原、がん抗原、およびこれらの成分等が挙げられる。好ましくは、がん抗原である。がん細胞を皮下に移植して腫瘍を形成させたマウスに対して本発明の核酸とがん抗原を併用投与したところ、腫瘍を顕著に退縮させることができたことから、本発明のワクチン組成物は、がんワクチンとして非常に有用であることが確認されている。本発明のワクチン組成物は、上記本発明のアジュバント組成物に抗原または抗原性成分を添加することにより製造することができる。また、本発明のワクチン組成物は、上記本発明のアジュバント組成物と同様に製剤化して実施することができる。
(1)配列番号3に示される塩基配列からなる一本鎖核酸Aと、配列番号4に示される一本鎖核酸Bにより形成されていることを特徴とする核酸。
(2)哺乳動物に対して、前記(1)に記載の核酸およびがん抗原を投与することを特徴とするがんの治療方法。
(3)がんの治療に使用するための前記(1)に記載の核酸。
(4)がん治療薬を製造するための前記(1)に記載の核酸使用。
(1)cM362−140(化学合成核酸)の作製
表1に記載の各一本鎖核酸(S1、S2、S3、AS1、AS2およびAS3)は、株式会社ジーンデザインに委託して合成した。RNA部分の化学合成にはtBDMS RNA amiditeを、DNA部分の化学合成には一般的なDNA amiditeを、S化(ホスホロチオエート)部分はPADSを使用した。合成方法は固相担体を用いたホスホロアミダイト法(Scaringe, S. A.et al, ;J Am Chem 1998;120:11820-11821)を基本とし、最適化したパラメーターを用いて実行した。合成完了後、一般的な方法を用いて塩基部分および2’位に存在する保護基を除去し、逆相HPLCにて精製を行った後脱塩し、各一本鎖核酸を得た。
cM362−139の構造を図2に、cM362−139を構成する核酸の塩基配列を表2に示す。
ODN-139 sense RNA (5’ primer)
5’-tgtaatacgactcactatagggaccagacaaagctggga-3’(配列番号19)
下線部はT7プロモーター配列
ODN-139 sense RNA (3’ primer)
5’-ggatacagtgccctgattaa-3’(配列番号20)
ODN-139 antisense RNA (5’ primer)
5’-tgtaatacgactcactataggatacagtgccctgattaa-3’(配列番号21)
下線部はT7プロモーター配列
ODN-139 antisense RNA (3’ primer)
5’-ccgtggtcatgctccgggaccagacaaagctggga-3’(配列番号22)
(1)血清を添加したPBS中の安定性
cM362−140およびcM362−139をそれぞれ、血清を含まないPBS、10%熱不活化FBS(ウシ胎児血清)、10%MS(マウス血清)または10%HS(ヒト血清)を含むPBSに20μg/mLの濃度で溶解し、37℃または42℃で60分間インキュベートした。インキュベート開始前(0分、血清を含まないPBSのみ)、5分後、15分後、30分後および60分後に、0.1μgの核酸が含まれるように溶液を取り、10×ローディングダイ(タカラバイオ)と混合し、臭化エチジウムを含む4%アガロースゲル(Nusieve 3:1 Agarose, Ronza)で電気泳動した。
結果を図3に示した。(A)がcM362−140の結果、(B)がcM362−139の結果である。37℃30分のインキュベーションでは、どちらの核酸も安定であったが、42℃30分のインキュベーションでは、インビトロ転写により作製したcM362−139は、血清(FBS、MS、HS)を含むPBS中で少し分解した。
PBSに代えてRNaseフリー水を用い、電気泳動に3%アガロースゲルを用いたこと以外は上記と同じ条件で安定性を検討した。
結果を図4に示した。(A)がcM362−140の結果、(B)がcM362−139の結果である。cM362−140はいずれの条件下でも安定であったが、cM362−139は、血清(FBS、MS、HS)存在下では部分的な分解が見られた。
以上の結果から、cM362−139よりcM362−140のほうが安定性に優れていることが明らかになった。
HEK293細胞(8×105cells/well)を6穴培養プレートに播種した。ヒトTLR3発現ベクター(400ng/well)または空ベクター(400ng/well)を、レポータープラスミドp−125(400ng/well)および内部コントロールベクターのphRL−TK(20ng/well、Promega)と共に、Lipofectamine2000(Invitrogen)を用いてHEK293細胞にトランスフェクションした。ヒトIFN−βプロモーター(-125〜+19)を含むレポータープラスミドp−125は、東京大学の谷口博士から提供された。培地には、10%熱不活化ウシ胎児血清(FCS、Invitrogen)および抗生物質を含むダルベッコ改変イーグル培地(DMEM、Invitrogen)を用いた。
(A)ヒトTLR3発現HEK293細胞の培地に核酸を直接添加
(B)ヒトTLR3発現HEK293細胞の培地に核酸とDOTAPリポソーム型トランスフェクション試薬(Roche)を添加
(C)HEK293細胞の培地に核酸とLipofectamine2000(Invitrogen)を添加
ルシフェラーゼ活性の測定には、Dual−Luciferase Reporter Assay System(Promega)を用いた。上記(A)および(B)は核酸添加から6時間後に、上記(C)は核酸添加から24時間後に、それぞれルシフェラーゼ活性の測定を行った。
C57BL/6Jマウス(野生型:WT)およびTLR3ノックアウトマウス(TLR3KO、大阪大学の審良博士から提供された)からそれぞれ脾臓を摘出し、コラゲナーゼ処理を行った。フィルターを通過させ、溶血後、培地で洗浄し、anti−CD11c microbeadsを用いたMACS system(miltenyi biotech)でCD11c陽性細胞を単離し、脾臓DCとした。培地には、10%熱不活化ウシ胎児血清(FCS、Invitrogen)および抗生物質を含むダルベッコ改変イーグル培地(DMEM、Invitrogen)を用いた。なお、本明細書に記載の全ての動物実験は、北海道大学動物実験委員会が作成したガイドラインに従って実施された。
脾臓DCを5×105個/500μL培地/wellとなるように24穴プレートに分注し、核酸(cM362−140、cM362−139、ポリIC、またはdsRNA140)20μg/mLを(A)単独で、(B)DOTAPリポソーム型トランスフェクション試薬(Roche)と共に、または(C)Lipofectamine2000(Invitrogen)と共に添加して24時間培養した。24時間後、培養上精を回収し、TNF−α、IL−6およびIFN−βの産生量を測定した。TNF−αおよびIL−6の測定にはBD CBA Flex Set Systemを用いた。IFN−βの測定には、マウスIFN−β用ELISAキット(PBL Assay Science)を用いた。
野生型C57BL/6Jマウス(9週齢、メス)またはTLR3KOマウス(9週齢、メス)に、50μgのcM362−140、cM362−139またはポリICを腹腔内投与した。一群あたり3匹のマウスを用いた。各核酸溶液は、RNaseフリー水を用いて調製した。核酸投与後、1時間目、3時間目および6時間目に尾静脈から採血し、血清中のTNF−α、IL−6およびIL−10を測定した。測定には、BD CBA Flex Set Systemを用いた。
(1)腫瘍退縮効果
C57BL/6Jマウスの背部を剃毛し、2×106cells/200μL(PBS)のEG7細胞(C57BL/6マウスの胸腺種に由来するEL4細胞にオボアルブミン抗原を発現させたがん細胞)を皮下に移植して腫瘍を形成させ、腫瘍体積(cm3)(長径×短径2×0.4)を経時的に測定した。移植後8日目(腫瘍体積:約0.6cm3)に、cM362−140単独、OVA単独、またはcM362−140+OVAを腫瘍周辺の皮下に投与した。対照としてPBS(−)のみを同様に投与した。一群あたり4匹のマウスを用いた。cM362−140の用量は50μg、OVAの用量は100μgとし、いずれもPBS(−)に溶解して調製した。投与容量は50μLとした。一回目の投与から7日後(移植後15日目)に二回目の投与を行った。OVAには、エンドトキシンフリーOVA(Hyglos)を用いた。
cM362−140のアジュバント活性を評価するために、EG7担がんマウスから脾臓細胞を調製し、テトラマーアッセイを行った。上記(1)において、PBS、cM362−140単独、OVA単独、またはcM362−140+OVAの二回目の投与から7日後(移植後22日目)に、定法に従い脾臓細胞を調製した。得られた脾臓細胞をFITC−CD8α(BioLegend)、PerCP/Cy5.5−7AAD(BD Biosciences)、APC−CD3ε(BioLegend)およびPE−OVA−テトラマー(MBL)で染色し、OVA特異的CD8陽性T細胞(テトラマー+/CD8+/CD3+細胞)を検出し、その割合を求めた。
上記(2)で調製した脾臓細胞(2×106cells/200μL/well)を96穴培養プレートに播種し、OVAペプチド(SL8)100nM存在下で3日間培養した後、培養上清中のIFN−γ量をBD CBA Flex Set Systemを用いて測定した。
結果を図10に示した。図10から明らかなように、cM362−140+OVA投与群は、他群と比較して、脾臓細胞のIFN−γ産生量が有意に増加していた(**:p<0.01)。なお、データは平均値±SEで示し、統計解析には一元配置分散分析およびボンフェローニテストを用いた。
実施例6の各結果から、cM362−140は抗原特異的細胞傷害性T細胞の増殖と活性化を誘導し、がん抗原と併用することにより優れたアジュバント効果を奏することが明らかになった。
C57BL/6−B16同系NK感受性腫瘍移植モデル(Akazawa T., T. Ebihara, M. Okuno, Y. Okuda, K. Tsujimura, T. Takahashi, M. Ikawa, M. Okabe, T. Ebihara, M, Shingai, N. Inoue, M. Tanaka-Okamoto, H. Ishizaki, J. Miyoshi, M. Matsumoto, and T. Seya. 2007. Antitumor NK activation induced by the Toll-like receptor3-TICAM-1 (TRIF) pathway in myeloid dendritic cells. Proc. Natl. Acad. Sci. USA. 104: 252-257.)を用いて、NK細胞の活性化による移植がんの退縮効果を評価した。C57BL/6Jマウス(野生型:WT)およびTICAM−1ノックアウトマウス(TICAM−1 KO、発明者らが作製)の背部を剃毛し、6×105cells/200μL(PBS)のB16メラノーマ細胞(B16D8)を皮下に移植して腫瘍を形成させ、腫瘍体積(cm3)(長径×短径2×0.4)を経時的に測定した。移植後12日目に、in vivo−JetPEIと混合した150μgのcM362−140または蒸留水(DW)を腫瘍周辺の皮下に投与した。一群あたり3匹のマウスを用いた。
Claims (5)
- 配列番号3に示される一本鎖核酸Aと、配列番号4に示される一本鎖核酸Bにより形成されている二本鎖核酸を含有することを特徴とするアジュバント組成物。
- 一本鎖核酸Aおよび一本鎖核酸Bのいずれの末端にもリン酸基が結合していないことを特徴とする請求項1に記載のアジュバント組成物。
- 一本鎖DNAを構成するヌクレオチドの全部又は一部がホスホロチオエート修飾されていることを特徴とする請求項1または2に記載のアジュバント組成物。
- 請求項1〜3のいずれかに記載のアジュバント組成物および抗原または抗原性成分を含むワクチン組成物。
- 前記抗原ががん抗原である請求項4に記載のワクチン組成物。
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