JP5820506B2 - フューリンノックダウン及びgm−csf増強(fang)癌ワクチン - Google Patents
フューリンノックダウン及びgm−csf増強(fang)癌ワクチン Download PDFInfo
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Description
TGF−βファミリーは、よく知られた免疫抑制活性を保持する。3種の公知のTGF−βリガンド(TGF−β1、β2及びβ3)は、ヒトの癌において偏在性である。TGF−β過剰発現は、腫瘍進行及び予後不良と相関する。腫瘍微小環境内におけるTGF−βレベルの上昇は、反応不顕性腫瘍応答とリンクする。TGF−βは、GM−CSF誘導性樹状細胞成熟化並びにそのMHCクラスII及び共刺激分子の発現を直接的及び間接的に阻害する。GM−CSF介在性免疫活性化におけるTGF−βのこのような負の影響は、GM−CSFに基づく癌細胞ワクチンにおけるTGF−β分泌を欠乏させることの理論的根拠を支持する。
、γIFNを分泌するとの証拠がある。局所的に作用するプロフェッショナルAPC[単球/マクロファージ、樹状細胞(DC, dendritic cell)]によるγIFN産生は、細胞の自己活性化及び近隣の細胞の活性化において重要となり得る。NK細胞と、場合によりプロフェッショナルAPCによるγIFN分泌は、感染症に対する初期の宿主防御において重要となる可能性があり、一方、Tリンパ球は、獲得免疫応答における主たるγIFN源となる。さらに、原発性及び移植腫瘍の発達の予防におけるγIFNの役割が同定された。γIFN産生は、APCによって分泌されるサイトカイン、中でも注目すべきはインターロイキン(IL)−12及びIL−18により制御される。γIFN産生の負の調節因子として、IL−4、IL−10、グルココルチコイド及びTGF−βが挙げられる。
iRNA(切断依存的)及び(切断非依存的RISCフォーマット型)miRNA又はmiRNA様いずれかのモチーフの両方を同時に組み入れた、二機能性となり得る。本発明の一実施形態において、shRNAは、RISC切断依存的及びRISC切断非依存的両方のフューリン発現阻害因子である。さらに、発現ベクターは、第一及び第二の核酸インサート間にインターカレートしたピコルナウイルス2Aリボソームスキップペプチドを含有することができ、プロモーターは、エンハンサー配列及びイントロンを含有し得るCMV哺乳類プロモーターとなることができる。二機能性shRNAの標的となるmRNA配列は、コード配列に限定されない。一実施形態において、shRNAは、フューリンmRNA転写産物の3’非翻訳領域(3’−UTR)配列を標的とすることができ、一実施形態において、フューリンmRNA転写産物のコード配列及び3’UTR配列の両方を同時に標的とすることができる。ワクチン作製に用いた細胞は、自家腫瘍細胞となることができるが、異種移植片増幅(expanded)自家腫瘍細胞、同種異系腫瘍細胞、異種移植片増幅同種異系腫瘍細胞又はそれらの組合せを用いてもよい。患者に投与したワクチン投薬量は、1×107細胞〜2.5×107細胞を含有する。FANGワクチンは、治療上有効量のγIFN(ガンマインターフェロン)と併せて与えることができる。γIFNの投薬量範囲は、50又は100μg/m2となり得る。
物は、メラノーマ、非小細胞肺癌、胆嚢癌、結腸直腸癌、乳癌、卵巣癌、肝癌、肝癌転移及びユーイング肉腫並びに他の患者由来のTGF−β産生癌からなる群から選択される癌の症状の予防、治療及び/又は寛解に用いられる。
記細胞(iv)懸濁液の生成が酵素による解体(enzymatic dissection)、機械的脱凝集又は両者によって達成されるステップと、(v)前記細胞懸濁液を電気穿孔することによって前記細胞を遺伝的に改変し、GM−CSF cDNAをコードする、プロモーターに作動可能に連結した第一の核酸インサート及びフューリンをコードするmRNA転写産物の領域とハイブリダイズして、これによりRNA干渉を介してフューリン発現を阻害できる1又は2以上の低分子ヘアピン型RNA(shRNA)をコードする、前記プロモーターに作動可能に連結した第二の核酸インサートを含む、bishRNAフューリン/GMCSF発現ベクタープラスミドを有するワクチンを作製するステップと、(vi)前記ワクチンを収集するステップと、(vii)前記ワクチンに放射線照射するステップと、(viii)
前記ワクチンを凍結するステップとを含む、フューリンノックダウン及び顆粒球マクロファージコロニー刺激因子(GM−CSF)増強(FANG)癌ワクチンを作製する方法について記載する。
た癌試料における癌細胞を遺伝的に改変するステップと、(iv)治療上有効用量の遺伝的に改変された細胞を前記対象に投与するステップとを含む。細胞のトランスフェクトに用いた発現ベクターは、2種の核酸インサートを含む。第一の核酸インサートは、GM−CSFをコードし、プロモーターに作動可能に連結する。第二の核酸インサートも該プロモーターに作動可能に連結し、フューリンをコードするmRNA転写産物の領域とハイブリダイズして、これによりRNA干渉を介してフューリン発現を阻害できる1又は2以上の低分子ヘアピン型RNA(shRNA)をコードする。本発明の一実施形態において、治療の標的となる癌は、ヒトメラノーマ又は非小細胞肺癌である。遺伝的に改変された細胞を増殖能力のない状態にするため、放射線照射してよい。FANGワクチンにおける遺伝的に改変された細胞は、自家細胞、同種異系細胞、異種移植片増幅細胞、樹立ヒト細胞系又はこれらの細胞型の組合せとなることができる。ワクチン接種のため、細胞は、月に1回を最大12用量、各回1×107細胞〜2.5×107細胞含有で対象に投与される。5×107までの用量増加は、安全であることが示された。
することが本明細書において立証されている。
基づく近年の臨床試験は、有望な結果をもたらした(Fakhrai, H., et al., Phase I clinical trial of a TGF-beta antisense-modified tumor cell vaccine in patients with
advanced glioma. Cancer Gene Ther, 2006. 13(12): p. 1052-60、Nemunaitis, J., GVAX (GMCSF gene modified tumor vaccine) in advanced stage non small cell lung cancer. J Control Release, 2003. 91(1-2): p. 225-31、Nemunaitis, J., et al., Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer. Cancer Gene Ther, 2006. 13(6): p. 555-62、Nemunaitis, J. and J. Nemunaitis, A review of vaccine clinical trials for non-small cell lung cancer. Expert Opin Biol Ther, 2007. 7(1): p. 89-102)。ホール癌細胞ワクチンは、定義済み及び未定義両方の腫瘍抗原に対し広範囲の多価免疫応答を潜在的に誘発し、これにより下方制御及び/又は抗原欠損バリアントの選択による腫瘍抵抗性の可能性に取り組むことができる(Ahmad, M., R.C. Rees, and S.A. Ali, Escape from immunotherapy: possible mechanisms that influence tumor regression/progression. Cancer Immunol Immunother, 2004. 53(10): p. 844-54、Hege, K.M., K. Jooss, and D. Pardoll, GM-CSF gene-modifed cancer cell immunotherapies: of mice and men. Int Rev Immunol, 2006. 25(5-6): p. 321-52)。
改変された腫瘍細胞が、他のサイトカインと比較して最も強力な抗腫瘍免疫の誘導を一貫して立証したことを示した(Dranoff, G., et al., Vaccination with irradiated tumor
cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A, 1993. 90(8): p. 3539-43)。サイトカイン導入遺伝子として組み込
まれると、GM−CSFは、自家又は樹立同種異系腫瘍細胞系のいずれかから得られた癌ワクチンペプチド、腫瘍細胞ライセート又はホール腫瘍細胞の提示を強化する(Hege, K.M. and D.P. Carbone, Lung cancer vaccines and gene therapy. Lung Cancer, 2003. 41 Suppl 1: p. S103-13)。GM−CSFは、造血前駆細胞のプロフェッショナル抗原提
示(APC)樹状細胞(DC)への分化を誘導し、それをワクチン接種部位へと誘引する(Dranoff, G., et al., Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A, 1993. 90(8): p. 3539-43、Huang, A.Y., et al., Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science, 1994. 264(5161): p. 961-5)。GM−CSFは、DC成熟化並びに腫瘍抗原捕捉、プロセシング及び提示の活性化
プロセスのアジュバントとしても機能し、それらの共刺激分子の発現並びにCD4+、CD8+T細胞、CD1d制限インバリアントナチュラルキラーT(NKT)細胞及び抗体産生B細胞の活性化のための二次リンパ組織への遊走能を上方調節する(Banchereau, J., et al., Immunobiology of dendritic cells. Annu Rev Immunol, 2000. 18: p. 767-811)。
クター及びT調節性細胞機能を変調するためのGVAXワクチン接種とそれに続く抗CTLA−4抗体の定期的な注入が、大多数の転移性メラノーマ患者において臨床上有意義な抗腫瘍免疫を生じ得ることを報告した。これらの知見は、GM−CSF増強自家癌ワクチンのワクチン接種が、特にFoxP3+Tregs活性を欠乏させ、腫瘍のMHCクラスIA鎖(MICA)発現を強化してこれによりナチュラルキラー(NK)及びT細胞を活性化し、セントラルメモリーT細胞CD4+及びCD8+応答を強化するアジュバント治療と併せた場合、免疫介在性腫瘍破壊の生成に成功したとの論文と一貫した。
ーリン基質BASE(β−部位APP切断酵素)、MT5−MMP及びBoc−RVRR−AMCの活性を完全に又は部分的にのいずれかで低下させる(Pearton, D.J., et al.,
Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates. Exp Dermatol, 2001. 10(3): p. 193-203)。本発明者らは、TGF−β1及びTGF−β2両方の活性が、CCL−247及びCRL−1585癌株において有意に低下することを特異的イムノアッセイによって見出し、TGF−βアイソフォーム発現におけるフューリン遮断の有効性を確認した。
S.A. Woodson, Self-splicing of a group I intron reveals partitioning of native and misfolded RNA populations in yeast. RNA, 2006. 12(12): p. 2149-59)。2種の
ステム−ループ二本鎖DNA配列を、DNAライゲーションにより10ピースの合成補完及び相互接続オリゴヌクレオチドと組み合わせて構築した。両端にBam HI部位を有する完成した241塩基対のDNAを、TAG発現ベクターのBam HI部位に、TGFβ2アンチセンス配列に代わって挿入した。shRNAインサート及び配向性のスクリーニング用に設計したPCRプライマー対により、挿入DNAの配向性をスクリーニングした。FANGコンストラクトは、カセット全体を駆動する単一の哺乳類プロモーター(CMV)を有し、GM−CSF及びフューリン二機能性shRNA転写産物の間に2Aリボソームスキップペプチドが介入し、ウサギポリAテールが続く。GM−CSF転写産物の終端に終止コドンが配置される。mRNAへのピコルナウイルス2A配列の挿入により、リボソームは、2A及び下流配列の接合部においてペプチド結合の形成をスキップし、単一のオープンリーディングフレームから2種のタンパク質を産生させる(Funston, G.M., et al., Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping. J Gen Virol, 2008. 89(Pt 2): p. 389-96)。しかし、shRNA又はアンチセンスが(具体例として)第二の転写産物として発現している場合、第一の転写産物のみ翻訳される。本発明者らは、2Aリンカーが、TAGワクチンによるほぼ等レベルのGM−CSF及び抗TFG−β転写産物の作製に有効であることを見出し、これを選出してFANGの同一設計を用いた。
クス(ECM))選択的装飾(標的)ステルス2層陥入リポソーム(stealthed bilamellar invaginated liposome)(BIV)を介した全身送達、(2)ヒトTGFβ1、TG
Fβ2、TGFβ3、IGF−II、IGF−1R、PDGF A及び一部の腫瘍型の場合はMT1−MMPを包含するが、これらに限定されないフューリン標的分子の腫瘍促進/維持効果を直接的に壊滅させるための、腫瘍選択的装飾(標的)ステルス型2層陥入リポソーム(BIV)を介した全身送達、(3)推定癌幹細胞におけるNOTCH/p300経路を直接的に壊滅させるための、腫瘍選択的装飾(標的)ステルス型2層陥入リポソーム(BIV)を介した全身送達、(4)炭疽、志賀(Shiga)、ジフテリア、破傷風、ボツリヌス症並びにエボラ及びマールブルグウイルスに関連する毒素の活性化を阻害するための、腫瘍選択的装飾(標的)ステルス型2層陥入リポソーム(BIV)を介した全身送達及び/又は(5)シュードモナス(Pseudomonas)介在性罹患率及び死亡率のリスク
が高まった疾患、例えば嚢胞性線維症の患者における抗生物質療法の補助としてのシュードモナス外毒素A産生を阻害するための、2層陥入リポソーム(BIV)(±装飾及び可逆的マスキング/ステルス化)の全身性及び/又は吸入送達。
量関連生存改善を立証した。後期(IIIB/IV)患者の2年間の生存は、2.5×10
7細胞/注射を超える投与を受けた患者に対し52%であり、これは、2年間で10%未満の生存である類似の患者病歴データと有利に比較される。試験患者はまた、サイトカイン産生(IFN−γ、p=0.006;IL−6、p=0.004;IL4、p=0.007)及びワクチンHLA抗原に対する抗体価(p=0.014)の有意な上昇を提示し、この結果は免疫活性化治療成績を示唆した(Nemunaitis, J., et al., Phase II trial
of Belagenpumatucel-L, a TGF-beta2 antisense gene modified allogeneic tumor vaccine in advanced non small cell lung cancer (NSCLC) patients. Cancer Gene Ther, 2009. 16(8): p. 620-4)。
vaccine in advanced cancer. Mol Therapy, 2009. 17 (Suppl 1): p. S206、Maples, P.B., et al. Autologous Tumor Cell Vaccine Genetically Modified To Express GM-CSF
and Block Expression of TGFb2 (Abstract # 553). in The Twelfth Annual Meeting of the American Society of Gene Therapy. 2009. San Diego, California)。療法に関
しグレード3毒性効果は観察されなかった。17名評定可能な患者のうち11名(65%)は、安定的な疾患を少なくとも3カ月間維持した。1名の患者は、イメージング判定基準によりCRを達成した(図4;メラノーマ)。よって、TAGワクチンは安全であると思われ、臨床有効性の証拠を有する。
ーゼと同様に、PCは、N末端プロセグメントが伸長した不活性型酵素原として合成され、このセグメントは小胞体において自己触媒的に除去されて機能性を得る(Khatib, A.M., et al., Proprotein convertases in tumor progression and malignancy: novel targets in cancer therapy. Am J Pathol, 2002. 160(6): p. 1921-35)。
108(3): p. 983-5)。以前に記載された腫瘍分泌性TGF−βの免疫抑制活性とは別に
、Tリンパ球における内在性発現フューリンのコンディショナルな欠失は、正常なT細胞を発達させるが、調節性及びエフェクターT細胞の機能を損ない、これらはTGF−β1の産生が少なかったことが見出された。フューリン欠損Tregは、T細胞伝達結腸炎モデルにおいて保護性が低く、正常なT細胞においてFoxp3を誘導できない。その上、フューリン欠損エフェクター細胞は、本質的に過活動(over-active)であり、野生型調
節性T細胞(Treg cell)の抑制活性に対し抵抗性である。APCにおいて、トランスゴルジ区画における細胞傷害性Tリンパ球感受性エピトープは、フューリン及び頻度が低いTAP非依存的経路によってプロセシングされる(Lu, J., et al., TAP-independent presentation of CTL epitopes by Trojan antigens. J Immunol, 2001. 166(12): p. 7063-71)。よって、T細胞によるフューリン発現は、末梢性免疫寛容の維持に不可欠であると思われ、これは、少なくとも一部には、TGF−β1産生調節におけるその非重複性必須機能のためである。
mRNA検出を図7に示す。トランスフェクション後、全ワクチンにおいてGMCSF
mRNAを検出したが、数値は持続的な問題であるmRNA品質に応じて可変的である。表1は、2種のFANGワクチンから得られた代表的なデータを例示する(図8及び9)。全試料を、正規化電気穿孔前Ct値マイナス正規化電気穿孔後放射線照射後Ct値として算出して(前−後)、デルタCt(ΔCt)を算出した。算出された0.00未満のΔCtは鋳型DNAの減少を表し、算出された0.00を超えるΔCtは鋳型DNAの増加を表す。ΔCt値を用いて、発現のパーセント変化を推算する(%発現)。100%未満の数値はDNAの(前から後の)減少を表し、100%を超える数値はDNAの(前から後の)増加を表す。shRNA/siRNAサイレンシングの性質は、鋳型DNAを最適に90%低下させることができ、これはΔCt=−3.3に相当する。(ΔCt=−1.0は、50%ノックダウンに相当する。)従って、下のデータは、FANGプラスミドDNAが、内在性フューリンを80〜26%(平均=48%)下に低下させることができ、下流標的のTGFβ1及びTGFβ2が、98〜30%(平均=75%)下に低下されることを立証する。フューリン二機能性shRNAの作用機構は、転写後及び翻訳レベルでフューリンタンパク質産生を遮断することである。フューリンタンパク質のレベル低下も、TGFβ1及びTGFβ2 mRNAの発現、TGFβ1及びTGFβ2タンパク質プロ型(proform)のそれらそれぞれのタンパク質の成熟(活性)型への変換に影響を与
え(フィードバック調節により)(Burghardt, I., et al., Pirfenidone inhibits TGF-beta expression in malignant glioma cells. Biochem Biophys Res Commun, 2007. 354(2): p. 542-7)、TGFβ→フューリン増幅ループに干渉することにより、フューリン
自体の発現をさらに鈍らせる(McMahon, S., M.H. Laprise, and C.M. Dubois, Alternative pathway for the role of furin in tumor cell invasion process. Enhanced MMP-2 levels through bioactive TGFbeta. Exp Cell Res, 2003. 291(2): p. 326-39)。TGFタンパク質プロ型の蓄積が、そのTGF遺伝子の転写をフィードバック阻害し得る可能性もある。
mRNA配列において予想されるsiRNA標的部位(図4B)を決定した。適格な翻訳及び3’UTR部位を標的とするsiRNA(図4B)を検査した。フューリンmRNAのFANGプラスミドDNAノックダウンの立証を図8及び9に示す。これはワクチンのうち2種しか検出できないが、その理由としては、容易に検出可能なフューリンmRNAはトランスフェクション前のこれら2種の腫瘍のみに存在したためである。bi−shRNAフューリンの作用機構は、転写後及び翻訳レベルにおけるフューリンタンパク質産生の遮断である。フューリンタンパク質のレベル低下も、TGF−β1及びTGF−β2 mRNAの発現、プロ型TGF−β1及びTGF−β2タンパク質のそれらそれぞれのタンパク質の成熟(活性)型への変換に影響を与え(フィードバック調節により)(Burghardt, I., et al., Pirfenidone inhibits TGF-beta expression in malignant glioma cells. Biochem Biophys Res Commun, 2007. 354(2): p. 542-7)、TGF−β→フューリ
ンループに干渉することにより、フューリン自体の発現をさらに鈍らせる(McMahon, S.,
M.H. Laprise, and C.M. Dubois, Alternative pathway for the role of furin in tumor cell invasion process. Enhanced MMP-2 levels through bioactive TGFbeta. Exp Cell Res, 2003. 291(2): p. 326-39)。TGFタンパク質のプロ型の蓄積がそのTGF遺伝子の転写をフィードバック及び阻害し得る可能性は、決して本発明を限定するものとして解釈されるべきではない。FANGの標的有効性の拡大は、自家ワクチンのTAG(TAG−004)及びFANG(FANG−004)両方のバージョンの作製に妥当な腫瘍組織を有する1名の患者(NSCLC)において最もよく立証される。FANG標本(FANG−004)を用いて、初濃度1840pg/mlから検出可能レベルを下回るようにTGF−β1(とTGF−β2)を欠乏させた。TGF−β2欠乏が予想されるにもかかわらず、この高レベルのTGF−β1は、TAG標本(TAG−004)によって変化しなかった(図1A〜1C及び図2A〜2F)。これらの知見は、FANGワクチン標本の機構的利点を支持する。
P.B., Senzer, N., Kumar, P., Wang, Z., papper, B.O., Yu, Y., Haddock, C., Tong,
A., Nemunaitis, J., Bi-functional shRNA: A novel approach of RNA interference.
(submitted), 2009)。siRNA成分は、ヘアピンとしてコードされ、パッセンジャー及びガイド鎖の完全にマッチする配列を網羅する。RNase H様活性を有するエンドヌクレアーゼであるRNA誘導サイレンシング複合体(RISC)のアルゴノート−2(Ago2)によるパッセンジャー鎖の切断後、ガイド鎖は、相補的標的mRNA配列と結合し、切断する。区別すると、「二機能性」ベクターのmiRNA様成分は、より低い熱力学的安定性を達成するため、コードshRNAヘアピン内のパッセンジャー及びガイド鎖間にミスマッチが組み入れられている。この立体配置は、Ago2非依存的に切断なし(切断非依存的プロセス)でRISCからパッセンジャー鎖を解離させ(Matranga, C., et al., Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell, 2005. 123(4): p. 607-20、Leuschner, P.J., et al., Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep, 2006. 7(3): p. 314-20)、翻訳阻止、mRNA分解及び部分的に相補的な標
的mRNAの細胞質プロセシングボディ(P−ボディ)における隔離により、miRNAガイド成分にその標的を下方制御させる。
Sci U S A, 2008. 105(23): p. 7964-9)。多部位標的化は、「シード配列」誘導オフターゲット効果の確率を増加させ得るため、本発明者らは単一部位に焦点を置く(Jackson,
S.A., S. Koduvayur, and S.A. Woodson, Self-splicing of a group I intron reveals
partitioning of native and misfolded RNA populations in yeast. RNA, 2006. 12(12): p. 2149-59)。
を確認した。siRNAの知見に基づき、3種の二機能性shRNAを構築した。最適な標的配列を同定した。
せて細胞懸濁液を生成し、次に洗浄してデブリを除去した。腫瘍細胞を計数した後、QCアリコートを採取して残りの細胞にFANGプラスミドを電気穿孔し、一晩インキュベートしてベクター導入遺伝子を発現させる。細胞を収集し、ガンマ線照射して細胞成長を停止させ、次に最後のQCアリコートの除去及びワクチン管理率凍結前に計数した。2日間の製造プロセスに続き、ほぼ3週間のQC検査フェーズを行い、その後、患者治療のためにワクチンを解禁する前に全ワクチンアッセイデータを評定する。FANG製造を行った9名の最初の患者は全員、全QC検査判定基準を満たした。
GFβ1)。TGFβ2の最小検出可能含量は、約7pg/mlである(R&D Systems社
製、QuantikineヒトTGFβ2)。GMCSFの最小検出可能含量は、約3pg/mlである(R&D Systems社製、QuantikineヒトGMCSF)。
セイを行った。トランスフェクション前試料(4×106細胞)を1日目に採取した。製造が完成した後、細胞培養物をELISA用試料の作製に設定できるように製造施設から試料を移動した。2日目に、トランスフェクション後、放射線照射後、凍結前試料(4×106細胞)を採取した。製造が完成した後、細胞培養物をELISA用試料の作製に設定できるように製造施設から試料を移動した。
現に干渉する誘発された抗体の発達を予防し、3)同時に発生するフューリン、TGFβ1及びTGFβ2の抑制は、GMCSF誘導樹状細胞成熟化の腫瘍関連阻害を最小化し得るため、記述されている製造プロセス間のGMCSF発現レベルの可変性に加えて、FANGワクチンにより達成された発現レベルは、臨床的に関連性があると判断される(Montenegro, D.E., et al., TGFbeta inhibits GM-CSF-induced phosphorylation of ERK and MEK in human myeloid leukaemia cell lines via inhibition of phosphatidylinositol 3-kinase (PI3-k). Cell Prolif, 2009. 42(1): p. 1-9 )。
−CSF及びTGF−βの成長調節効果をインビトロ代用モデルとして用いて、FANGワクチン培養上清の調製におけるサイトカイン生物活性を検証した。
、必要であれば、混合型(biphenotypic)B骨髄単球性白血病性CD10+CD15+MV4−11細胞により確認した(Santoli, D., et al., Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-1 alpha, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines. J Immunol, 1987. 139(10): p. 3348-54)(ATCC、メリーランド州ロックビル)。これらの細胞系は両者共に、ng/ml量におけるGM−CSFの正の増殖効果及びTGF−βの負の阻害活性に対する応答を示した(Montenegro, D.E., et al., TGFbeta inhibits GM-CSF-induced phosphorylation of ERK and MEK in human myeloid leukaemia cell lines via inhibition of phosphatidylinositol 3-kinase (PI3-k). Cell Prolif, 2009. 42(1): p. 1-9 )。増殖活性は、Easycount Viasureアッセイ(Immunicon社)及びMTTアッセイにより決定されるであろう(Tong, A.W., et al., Intratumoral injection of INGN 241, a nonreplicating adenovector expressing the melanoma-differentiation associated gene-7 (mda-7/IL24): biologic outcome in advanced cancer patients. Mol Ther, 2005. 11(1): p. 160-72)。
いた質量分析解析のための調製において、Opti-TOF(商標)LC/MALDI挿入(123×81mm)プレート(Applied Biosystems社)上に画分をスポットした。次に、GeneGo、MetaCoreソフトウェア一式によりタンパク質及び遺伝子発現データの両方を評定した。
原及び共刺激分子(CD80/86)発現、4)FANGトランスフェクション後に2倍以上ディファレンシャルに発現する上述のカテゴリーとは無関係のタンパク質に特に注意を払った。
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Claims (5)
- プロモーター及び前記プロモーターに作動可能に連結した核酸インサートを含む発現ベクターを含むトランスフォーミング増殖因子ベータ(TGF−ベータ又はTGF−β)の発現阻害剤であって、前記インサートが、フューリンをコードするmRNA転写産物の領域とハイブリダイズして、これによりRNA干渉を介してフューリン発現を阻害できる1又は2以上の二機能性低分子ヘアピン型RNA(shRNA)をコードし、
前記shRNAが、切断依存的及び切断非依存的の両方であるフューリン発現阻害因子であり、
前記shRNAが、フューリンmRNAの核酸300〜318、731〜740、1967〜1991、2425〜2444、2827〜2851、又は2834〜2852に向けられる、
前記発現阻害剤。 - ピコルナウイルス2Aリボソームスキップペプチド配列をコードするヌクレオチド配列が第一及び第二の核酸インサート間にインターカレートされている、請求項1に記載の発現阻害剤。
- プロモーターが、CMV哺乳類プロモーターである、請求項1に記載の発現阻害剤。
- CMV哺乳類プロモーターが、CMV IE 5’UTRエンハンサー配列及びCMV IEイントロンAを含む、請求項3に記載の発現阻害剤。
- 二機能性低分子ヘアピン型RNAが、配列番号1を含む、請求項1に記載の発現阻害剤。
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