JP4394570B2 - 抗癌活性を有するHer−2/neuDNAワクチン - Google Patents
抗癌活性を有するHer−2/neuDNAワクチン Download PDFInfo
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- JP4394570B2 JP4394570B2 JP2004521272A JP2004521272A JP4394570B2 JP 4394570 B2 JP4394570 B2 JP 4394570B2 JP 2004521272 A JP2004521272 A JP 2004521272A JP 2004521272 A JP2004521272 A JP 2004521272A JP 4394570 B2 JP4394570 B2 JP 4394570B2
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Description
本発明の他の目的は、前記プラスミドコンストラクトおよび薬剤学的に許容可能な担体を有効成分とする、癌の予防および/または治療用DNAワクチン組成物を提供することである。
本発明のまた他の目的は、有効量の前記DNAワクチンを投与する段階を含む、癌を予防および/または治療するための方法を提供することである。
まず、ヒトHer−2/neu全長をコーディングするプラスミドは該プラスミドDNAが導入された細胞の生理機能に悪影響を及ぼし得るため、本発明は細胞質キナーゼドメイン(細胞内ドメイン)が除去された、短縮Her−2/neu遺伝子をコーディングするHer−2/neu発現プラスミドコンストラクトを提供する。この短縮ヒトHer−2/neu遺伝子はHer−2/neu膜貫通および細胞外ドメインを含む配列番号:2の塩基配列を有し、外来遺伝子を非常に高い効率で発現するpTV2ベクター(Lee, S. W. et. al., J. Virol. 72: 8430-8436, 1998)に挿入される。
Her−2/neuを発現するヒト乳房癌腫SK−BR3細胞株(ATCC HTB−30)とラットの結腸腺癌腫細胞株であるCT26(ATCC CRL−2639)はATCC(American Type Culture Collection, Manassas, VA, USA)から入手した。ヒト乳房癌細胞株SK−BR3細胞は10%熱不活性化ウシ胎児血清(FBS, GIBCO, Gaithersburg, MD)と1%ペニシリン−ストレプトマイシン(GIBCO)を含有するRPMI1640培地(Bio Whittaker, Walkersvile, MD)で培養した。Her−2/neuを発現する移入細胞Her2−CT26細胞株はヒトHer−2/neuをコードするcDNA(NCBI:M11730)をCT26細胞に形質導入して製造した。Her2−CT26とCT26細胞は10%熱不活性化ウシ胎児血清と1%ペニシリン−ストレプトマイシンを含有するIMDM(BioWhittaker)培地で培養した。
プラスミドpNeuTM,pNeuECD,pNeuTM−gDs,pNeuECD−gDs,pCKTM,pCKECD,および対照群ベクターpTV2とpCKで各々形質転換された大腸菌DH5α(Escherichia coli strain DH5α)をLB液体培地(Difco, Detroit, MI)で培養した。培養した大腸菌形質転換体からエンドフリーキアゲンプラスミド−ギガキット(Endofree Qiagen Plamid-Giga Kits, Qiagen, Chatsworth, CA)を用いるアルカリ溶解方法によって、製造社の指針に従ってプラスミドDNAを大量分離した。このように分離したDNAを沈澱させた後、滅菌PBS(Bio Whittaker)に2mg/mlの濃度で懸濁し、免疫接種スケジュールに従って使用時まで−20℃で保管した。
血清がHer−2/neu表面タンパク質に特異的に反応するかどうかを調査するために、SK−BR3、Her2−CT26およびCT26細胞をセルスクレーパー(cell scraper, Nunc, Naperville, IL)を用いて培養フラスコから遊離させ回収した。回収した細胞を2%ウシ胎児血清および0.1%アジ化ナトリウムを含有するRPMI1640培地からなるFACS緩衝溶液で洗浄した。分析当り約2×105個の細胞を血清または対照群抗体の連続希釈溶液とともに4℃で30分間反応させた。培養した細胞をさらに同じFACS緩衝溶液で3回洗浄した後、マウスIgGに特異的なFITC−接合ヤギモノクローナル抗体(Sigma)と4℃で30分間反応させて染色した。染色された細胞を2回洗浄した後、同じFACS緩衝溶液に再懸濁した。死んだ細胞をデータから排除するために、細胞懸濁液に1μg/mlヨウ化プロピジウム(Sigma)を入れて分析前に30秒間培養した。ヨウ化プロピジウム染色によって陰性と判定された細胞のみを分離し、さらに腫瘍細胞に対する結合分析に用いた。フローサイトメトリー分析はPAS IIIiフローサイトメトリー分析器(Partec GmbH, Munster, Germany)を用いて行った。
約1×104個のSK−BR3細胞を1mg/mlポリ−L−リジンがコーティングされたLab−Tekチャンバカバーガラス(Nunc, Napervile, IL)で3日間培養した。この細胞を4%パラホルムアルデヒドを含有するPBS緩衝溶液で室温で10分間処理して固定し、DMEM培地で3回洗浄した後、1%ヤギγ−グロブリンを含有するDMEM培地で4℃で1時間遮断した。遮断溶液中で1:50で希釈されたマウス血清を前記溶液に添加し、4℃で8時間培養した後、洗浄し、R−フィコエリスリン−接合ヤギ抗マウス免疫グロブリン2次抗体(Southern Biotech, Birmingham, AL)と室温で30分間反応させた。スライドをゲル/マウント培地(Gel/Mount media, Fisher)に載せた後、共焦点走査顕微鏡(Leica TCS-SP laser scanning microscopy)で観察した。
簡略に説明すれば、各々のマウスに滅菌PBS100μlに溶解したプラスミドDNA100μgを前脛骨筋内に筋肉注射した。注射部位はブピバカイン−HCl(bupivacaine-HCl, ASTRA, Westborough, MA)で予め処理した。治療用ワクチンに対する毎日の免疫化のために、ブピバカイン−HClを一番目の免疫化の直前に1回のみ前処理した。血清は定められた時間に眼窩の後部の叢(retro-orbital plexus)から取って抗Her−2/neu抗体が存在するかどうかを測定した。
免疫されたマウスから脾臓を切り出して得られた脾臓細胞をマイトマイシンC(Sigma)処理されたHer2−CT26細胞と6日間培養し、4時間51Cr−放出分析でCT26またはHer2−CT26標的細胞の溶解に対して分析した。
<反応式1>
特異的溶解率(%)=100×[(cpm実験群−cpm自然溶解群)/(cpm最大溶解群−cpm自然溶解群)]
滅菌PBSに懸濁したHer2−CT26細胞をわき腹に皮下注射するか、静脈注射してマウスを接種した。各腫瘍の3次元的サイズをカリパス(caliper)を用いて測定し、体積を下記反応式2によって計算した:
<反応式2>
腫瘍体積(mm3)=(幅×長さ×厚さ)mm3×π/6
pTV2およびpTV2−gDs(Lee, S. W. et al., J. Virol. 72:8430-8436, 1998)およびpCK(Lee Y., et. al., Biochem Biophys Res Commun. 272:230-235, 2000; Deposit Accession No: KCCM-10179)を発現ベクターとして使用した。pTV2−gDsはヘルペスシンプレックスウイルスI型糖タンパク質Dの信号配列が発現ベクターpTV2にクローニングされた発現ベクターである。全ヒトHer−2/neu遺伝子(配列番号:1)をコーディングするcDNAをpRC/CMV骨格(Invitrogen, San Diego, CA)に挿入して全長のHer−2/neuプラスミド(9.6Kb)を製造した。
様々なpNeuプラスミドコンストラクトが抗−Her−2/neu抗体を誘導できるかどうかを次のような方法で実験した。
pNeuコンストラクトの免疫接種によって免疫化されたマウスにおいてHer−2/neu特異的な抗体反応の高さと低さ(図2)を立証するために、同じマウスで誘導されたHer−2/neu特異的なCTL反応を次のような方法で評価した。
Her−2/neuを発現する同一種起源のマウス腫瘍細胞株であるHer2−CT26に対する抗癌免疫効果を下記のように測定した。
実施例2〜4は、互いに異なるpNeuプラスミドで免疫化されたマウスにおいてHer−2/neu−特異的な抗体力価は相反する結果を示すが、CTL反応は互いに類似することを立証した。また、pNeuTM,pNeuECD,pNeuTM−gDs,またはpNeuECD−gDsで免疫化された各群のすべてのマウスは5×104皮下注射腫瘍試験感染を克服した。皮下注射または静脈注射によってマウスに注入された腫瘍細胞の数が免疫化されたマウスにおいて腫瘍を誘導するにはあまりにも少なかったため、互いに異なるpNeuコンストラクトによって誘導された免疫反応における相異による抗腫瘍効果を区別することが難しかった。そこで、本実験では、Her2−CT26の抑制のためのHer−2/neu−特異的な抗体およびCTL反応の相対的な重要性を評価するために、一番目の腫瘍実験に使用された腫瘍細胞の数よりも皮下注射する腫瘍細胞の数を100倍(5×106個/マウス)増加させ、静脈注射する腫瘍細胞の数を40倍(2×106個/マウス)増加させた。静脈注射は静脈に投与された癌細胞数が過度な場合、血管閉鎖が起こるおそれがあるので、細胞の数を2×106個以上は使用することができなかった。比較のため、4つの互いに異なるHer−2/neu発現プラスミドのうちHer−2/neu特異的な抗体力価において最も大差を示すpNeuECDとpNeuECD−gDsを選定した。マウスは同一な免疫接種スケジュール(図1b)に従ってプラスミドDNA100μgを3回筋肉注射で接種し、プラスミドDNAを3次注射してから10日後、各マウスにHer2−CT26細胞を5×106個皮下注射するか、2×106個静脈注射した。
予防モデル腫瘍実験を免疫化されたマウスに腫瘍細胞を試験感染させて行った。治療モデルにおいてpNeuECDとpNeuECD−gDsの抗腫瘍効果を比較するために、マウスをまず腫瘍細胞で試験感染させた後、プラスミドDNAを筋肉注射で投与した。6週齢のマウスに1×105個または5×105個のHer−2/neu細胞を静脈注射した後、4群に分けた。腫瘍細胞を接種してから1時間後、各々のマウスにpNeuECDまたはpNeuECD−gDs 100μgをまず筋肉注射し、同じプラスミドDNAを4日間毎日筋肉注射で接種した。
ワクチンの臨床的効能を増強させるために、Her−2/neu DNAプラスミドベクターをpTV2よりも強力なプロモーター活性を有するpCKベクターをもって製作した。pNeuECDおよびpNeuTMから得られた短縮Her−2/neu遺伝子のKpnI−XbaI切片をpCKベクターのKpnI−XbaI制限酵素部位に挿入した。これから、Her−2/neuの細胞外および膜貫通ドメインを発現するpCKTMおよびHer−2/neuの細胞外ドメインを発現するpCKECDを製作した。
Her−2/neuのpCKコンストラクトの抗腫瘍効果を決定するために、雌BALB/cマウスに100μgのPBS,pCK,pCKECDまたはpCKTMを2週間隔で3回ずつ筋肉注射して免疫接種した。最終の免疫接種後前記マウスに1×106 Her2−CT26を皮下注射または静脈注射によって試験感染させた。Her2−CT26の皮下注射によって誘導された成長した固形腫瘍の3次元的サイズをカリパスを用いて測定した。生存したマウスの数を毎日数え、その結果を処理群当りの生存したマウスの百分率で示した。
Her−2/neu DNA免疫接種において分子的免疫増強剤としてサイトカイン遺伝子を用いるために、6つのサイトカイン遺伝子を含むpCKベクター、pCK−GMCSF,pCK−IL12,pCK−IL15,pCK−IL18,pCK−Eta1およびpCK−Flt3Lを下記のように製作した。抗原提示細胞の増殖および活性化を促進するGM−CSFおよびFlt3Lは、樹状細胞のような専門的な抗原提示細胞への伝達効率を向上させ、免疫反応を増加させることと期待される。IL−12,IL−15,IL−18およびEta−1は代表的なTH1 skewingサイトカインであって、癌免疫性に非常に重要な細胞−媒介性免疫反応を誘導することと期待される。
pCKTMおよびサイトカインプラスミドの併用投与によって誘導される抗腫瘍活性を決定するために、BALB/cマウスを用いて予防および治療実験を行った。図13Aおよび14Aに示すように、pCKTMと各々のサイトカインプラスミドで免疫接種する前と後にBALB/cマウスをHer2−CT26細胞で試験感染させた。100μgのpCKTMおよび100μgの各々のpCK−サイトカインプラスミドをBALB/cマウスに筋肉注射によって併用投与した。前記マウスを2次免疫接種し、3週間後1×106 Her2−CT26細胞で静脈注射または皮下注射によって試験感染させた。腫瘍成長は一週間に2回カリパスで測定し、体積は各マウスに対して測定した。
Her−2/neu DNA免疫接種の抗腫瘍活性を増加させるために、Her−2/neuタンパク質および各々のサイトカインが独立して転写されるバイシストロニック(bicistronic)プラスミドpCKTM−GMCSF,pCKTM−Flt3L,pCKTM−Eta1,pCKTM−IL12,pCKTM−IL15,pCKTM−IL18およびpCKTM−IL23を製作した。
pCKTM−サイトカインプラスミドの予防的抗腫瘍活性を評価するために、マウスを7つのpCKTM−サイトカインプラスミドの各々(pCKTM−GMCSF,pCKTM−Flt3L,pCKTM−Eta1,pCKTM−IL12,pCKTM−IL15,pCKTM−IL18およびpCKTM−IL23)で図15Bに示す免疫接種スケジュールに従って免疫化した。
Claims (3)
- pTV2またはpCKベクターに、ヒトHer−2/neuタンパク質の信号ペプチド、細胞外ドメイン、及び膜貫通ドメインからなるタンパク質をコーディングする配列番号:2の塩基配列により表されるHer−2/neu遺伝子及び顆粒球−マクロファージコロニー−刺激因子(granulocyte-macrophage colony-stimulating factor, GM-CSF)遺伝子を、これら遺伝子の間に内部リボソーム結合部位(IRES)を挟んでバイシストロニックに挿入して製造されたHer−2/neuプラスミドコンストラクトを含む癌治療剤。
- 前記Her−2/neuプラスミドコンストラクトが、pNeuTM(KCCM−10393)またはpCKTM(KCCM−10396)ベクターにGM-CSF遺伝子を挿入して製造されたものであることを特徴とする請求項1記載の癌治療剤。
- 請求項1記載のプラスミドコンストラクトを有効成分とし、薬剤学的に許容可能な担体を含む、癌を治療するためのDNAワクチン。
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US9956276B2 (en) | 2012-01-19 | 2018-05-01 | Duke University | Vaccines against antigens involved in therapy resistance and methods of using same |
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