TWI558412B - 包含陽離子性脂質之組合物及其用途 - Google Patents
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Description
本發明大體而言係關於刺激免疫反應,且更特定言之,係關於脂質在免疫反應中之作用。
本申請案主張美國申請案第12/049,957號(Weishu Chen, Weili Yan, Kenya Toney, Gregory Conn, Frank Bedu-Addo及Leaf Huang於2008年3月17日申請且名為"Stimulation of an Immune Response by Cationic Lipids")之權利,該申請案主張以下各美國臨時申請案之權利:第60/896,412號,Leaf Huang於2007年3月22日申請且名為"Cationic-Lipid Based Immune Stimulant for Treatment of Disease";第60/911,549號,Leaf Huang及Weishu Chen於2007年4月13日申請且名為"Cationic-Lipid Based Dual Function Delivery System and Immune Stimulant for Treatment of Disease";第60/948,512號,Leaf Huang, Weishu Chen及Weili Yan於2007年7月9日申請且名為"Cationic-Lipid Based Dual Function Delivery System and Immune Stimulant for Treatment of Disease";及第60/983,799號,Weishu Chen及Leaf Huang於2007年10月30日申請且名為"Induction of Immune Responses by a Liposomal Peptide Formulation",該等申請案之揭示內容以引用方式全文併入本文中。
本部分旨在介紹此技藝中可能與本發明之各種態樣相關
的各方資訊,茲描述且/或主張如下。咸信本論述有助於提供背景資訊,以利於更好地理解本發明之各種態樣。因此,應瞭解,該等陳述應從此角度判讀,而非視為對先前技術之認可。
對於全世界患者而言,開發供人類使用之安全且有效的免疫療法仍為緊迫的醫學需求。為誘發適當的免疫反應,可在疫苗設計或免疫療法中使用增強、引導或促進免疫反應的免疫調節劑(immunologic modifier/immunomodifier)[Gregoriadis, G.,Immunological adjuvants: a role for liposomes
. Immunol Today 11:89 (1990)]。舉例而言,疫苗可包含刺激免疫反應之抗原。然而,包含抗原的某些潛在疫苗僅為免疫反應之弱刺激劑,原因在於該等疫苗不能將抗原有效地傳遞至免疫系統之抗原呈現細胞("APC")且/或抗原具有弱免疫原性。因此需要可將抗原有效傳遞至APC並刺激免疫系統對抗原反應的免疫療法。免疫調節劑具有充當該免疫療法的潛在作用。該等免疫療法可具有該等及其他益處。舉例而言,當作為治療性疫苗之一部分而使用時,免疫調節劑應至少:(1)改良抗原之傳遞及/或在APC中之加工[Wang, R. F.及Wang, H. Y.Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells
. Nat Biotechnol 20:149 (2002)];(2)誘導免疫調節性細胞因子之產生,有利於形成針對疫苗抗原的免疫反應,從而促成細胞介導之免疫,包括細胞毒性T-淋巴細胞("CTL");(3)減少有效疫苗所需之免疫次數或抗原量
[Vogel, F. R.Improving vaccine performance with adjuvants
. Clin Infect Dis 30 Suppl 3:S266 (2000)];(4)增加疫苗抗原之生物或免疫半衰期;及(5)藉由抑制免疫抑制因子來克服針對抗原的免疫耐受性[Baecher-Allan, C.及Anderson, D. E.Immune regulation in tumor-bearing hosts
. Curr Opin Immunol 18:214 (2006)]。
目前,用於增強抗原(諸如肽或蛋白質抗原)誘發免疫反應功效的主要藥劑類別為佐劑,諸如油包水乳液、礬及其他增強抗原反應的化學藥劑;然而,該等佐劑不為如上所述的免疫調節劑,原因在於該等佐劑本身不具有直接的免疫調節作用[Vogel, F. R.及Powell, M. F.A compendium of vaccine adjuvants and excipients
, Pharm Biotechnol 6:141 (1995)]。數種該等佐劑可供用於動物且其中有些佐劑已經受臨床試驗測試。除諸如鋁鹽之傳統佐劑外,諸如流感病毒體[Gluck, R.及Walti, E. 2000.Biophysical validation of Epaxal Rerna, a hepatitis A vaccine adjuvanted with immunopotentiating reconstituted influenza virosomes (IRIV)
. Dev Biol (Basel) 103:189 (2000)]及Chiron之MF59[Kahn, J. O.等人,Clinical and immunologic responses to human immunodeficiency virus (HIV) type ISF2 gp120 subunit vaccine combined with MF59 adjuvant with or without muramyl tripeptide dipalmitoyl phosphatidylethanolamine in non-HIV-infected human volunteers
. J Infect Dis 170:1288 (1994)]之產品正在市場上銷售,該等產品具有固
有的免疫作用。舉例而言,基於亞微乳液之佐劑MF59被樹突狀細胞內在化[Dupuis, M.等人,Dendritic cells internalize vaccine adjuvant after intramuscular injection
. Cell Immunol 186:18 (1998)]。然而,根據有關HSV及流感疫苗的臨床試驗報告[Jones, C. A.及Cunningham, A. L.Vaccination strategies to prevent genital herpes and neonatal herpes simplex virus (HSV) disease
. Herpes 11:12 (2004); Minutello, M.等人,Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons
. Vaccine 17:99 (1999)],來自動物模型的證據表明MF59佐劑使中和抗體之產生增強、而非使T-細胞反應增強。因此,需要刺激細胞介導之免疫反應的新穎方法。
此外,如上所述,有些抗原為免疫反應之弱刺激劑。因此,除將抗原與如上所述之刺激免疫反應的物質共同投藥外,亦可對弱免疫原性抗原加以修飾以增強其免疫原性。舉例而言,弱免疫原性抗原可與免疫原性肽、多醣或脂質偶聯以增強其免疫原性。然而,弱免疫原性抗原與該等類型化合物的簡單偶聯可能不足以誘發免疫反應。舉例而言,所得免疫反應可針對偶聯化合物上而非弱抗原上的免疫原性抗原決定基,或偶聯抗原可能未有效地傳遞至免疫系統之APC。因此,需要其他方法刺激針對具有弱免疫原性之抗原的免疫反應。
以下闡述本發明之某些例示性態樣。應瞭解,該等態樣僅供向讀者提供本發明可能採取之某些形式之概要且該等態樣並非用以限定本發明之範圍。實際上,本發明可涵蓋下文可能未明確闡述的多種態樣。
本發明係關於使用陽離子性脂質(1)將抗原有效地呈現或傳遞至免疫系統及(2)刺激免疫系統響應抗原,該等陽離子性脂質在一定劑量及組成條件下可作為新穎類別的免疫刺激劑起作用。
脂質體已廣泛用於傳遞小分子量藥物、質體DNA、寡核苷酸、蛋白質及肽。與使用活減毒疫苗或病毒載體(諸如牛痘或流感病毒)的傳統免疫法相比,使用脂質體媒劑作為非病毒抗原載體的疫苗較佳。美國專利第7,303,881號描述了一種簡單卻有效的基於脂質之免疫療法,即由陽離子性脂質與抗原兩種分子組成的陽離子性脂質/抗原複合物,且可向該複合物中添加其他組分,諸如穩定劑、佐劑及表面調節劑[參見美國專利第7,303,881號]。在小鼠模型中,由陽離子性脂質與抗原[例如E7,一種人類乳突病毒("HPV")]組成的調配物誘導針對HPV-陽性TC-1腫瘤之預防性與治療性抗腫瘤免疫反應。結果證明與抗原複合之陽離子性脂質體可刺激免疫反應且引發樹突狀細胞(APC)與T-細胞之相互作用。
在本發明中,為進一步瞭解陽離子性脂質/抗原複合物誘導強免疫反應之能力,進行了其他研究,結果發現陽離
子性脂質在低劑量條件下、藉由使存在於所有哺乳動物種內之有絲分裂原活化蛋白質("MAP")激酶信號傳導途徑之組分活化可單獨充當強免疫活化劑。與抗原組合後,陽離子性脂質/抗原複合物在低劑量條件下誘導特異於該複合物中所調配之抗原的強免疫反應。在更高陽離子性脂質劑量下,於免疫細胞中產生過量的活性氧(ROS)且減弱所觀測到之免疫反應。
因此,本發明之一態樣提供具有至少一種陽離子性脂質的組合物,該陽離子性脂質之劑量足以藉由活化受檢者免疫系統之細胞之MAP激酶信號傳導來誘導受檢者之免疫反應。
本發明之另一態樣提供一種如下誘導受檢者之免疫反應的方法:藉由將陽離子性脂質投與受檢者來活化MAP激酶信號傳導途徑。
本發明之另一態樣提供具有至少一種陽離子性脂質的組合物,該陽離子性脂質之劑量足以藉由誘導受檢者免疫系統之細胞產生活性氧("ROS")來誘導免疫反應。陽離子性脂質複合物刺激ROS產生以達到足以使免疫反應強於在該至少一種陽離子性脂質不存在時所呈現之免疫反應之程度。
本發明之另一態樣提供一種如下誘導免疫反應的方法:將陽離子性脂質複合物投與受檢者以誘導受檢者免疫系統之細胞產生活性氧("ROS")。陽離子性脂質複合物刺激ROS產生以到達足以使免疫反應強於在該至少一種陽離子
性脂質不存在時所呈現之免疫反應之程度。
本發明之其他態樣包括添加至少一種抗原以形成陽離子性脂質/抗原複合物,在此情況下,免疫反應為抗原特異性反應。
參考附圖(在整個圖式中,相同標記代表相同部分)研讀以下實施方式,可更好地瞭解本發明之各種特徵、態樣及優點。
以下描述本發明之一或多個特定實施例。為提供該等實施例之簡要說明,本說明書中可能未描述現實實施例之全部特徵。應瞭解,在任何該現實實施例的開發中,為實現開發者之特定目標,須作出很多特定於實施例的決斷,決斷可因實施例不同而不同。此外,應瞭解,此開發工作可能複雜而耗時,但仍為例行程序,以確保一般熟習此項技術者自本揭示內容收益。
當介紹本發明之元件(例如其例示性實施例)時,冠詞"一"及"該"旨在意謂存在一或多個該等元件。術語"包含"、"包括"及"具有"意欲為內含性且意謂可能存在除所列元件外的其他元件。
本發明之一態樣提供使哺乳動物產生免疫反應以預防或治療疾病的陽離子性脂質。陽離子性脂質可藉由活化MAP激酶信號傳導途徑之各種組分、以劑量依賴性方式獨立地起免疫調節劑之作用,諸如用於產生趨化因子及/或細胞因子。不同哺乳動物種類中,所觀測到的最佳劑量範圍不
同。舉例而言,在齧齒動物種類中,最佳陽離子性脂質劑量可在50-300奈莫耳(nmole)之間。本文中所述的特定劑量及組成僅具有例示性且熟習此項技術者可判定適用於既定受檢者之劑量。在另一態樣中,可使低劑量範圍內之陽離子性脂質與抗原或藥物結合,以便呈現給免疫系統之細胞,同時刺激強抗原特異性免疫反應。在本發明之有些態樣中,抗原為脂肽(lipopeptide)。
美國專利第7,303,881號揭示,與疾病相關抗原複合的多種陽離子性脂質可刺激預防特定疾病(例如HPV陽性癌症)的預防性免疫反應以及殺死表現特定抗原之細胞且使疾病得到有效治療的治療性免疫反應。為進一步瞭解陽離子性脂質之免疫刺激能力,目前已使用屬於廣泛脂質類別的DOTAP、DOEPC及氯化丙基-N,N,N-三甲基銨("DOTMA")三種陽離子性脂質進行研究,在以上引用專利中已證明其可起免疫刺激劑之作用。由該等研究發現,陽離子性脂質在某些低劑量範圍內可獨立地作為免疫調節劑或組合物,以經(或不經)抗原刺激免疫反應。當將陽離子性脂質與抗原複合時,產生抗原特異性免疫反應。
在另一態樣中,在上述某些劑量條件下所投與的本發明之陽離子性脂質組合物在將抗原傳遞至免疫系統之細胞的同時可刺激對MAP激酶信號傳導途徑之各種組分之誘導,並活化身體對抗疾病的免疫反應。如下文實例所證明,陽離子性脂質以劑量依賴性方式誘導活性氧("ROS")之產生。然而,超出陽離子性脂質之最佳劑量時,高ROS產生
誘導免疫系統細胞之細胞凋亡,使脂質產生強免疫反應的能力減弱。特定範圍之ROS之產生又引起細胞因子及趨化因子之產生,以調節免疫反應。因此,陽離子性脂質之最佳劑量為有效刺激ROS產生以達到足以刺激免疫反應強於在陽離子性脂質不存在時所呈現的免疫反應之程度(同時不刺激足以誘導免疫系統細胞發生大量細胞凋亡,亦即,減弱免疫反應之足夠細胞凋亡的過量ROS產生)並活化MAP激酶信號傳導途徑的量。如上所述,最佳劑量可隨物種而變且易由一般熟習此項技術者判定。
在又一態樣中,將最佳劑量之陽離子性脂質與一或多種抗原組合投藥。在此情況下,陽離子性脂質/抗原組合能夠產生特異於與陽離子性脂質組合傳遞之抗原的免疫反應。所產生的反應可包括特異性細胞毒性T細胞、記憶T細胞或B細胞之產生,從而預防與抗原相關之特定疾病或形成針對與抗原相關之特定疾病的治療性反應。
本發明之陽離子性脂質可為陽離子性脂質複合物之形式。陽離子性脂質複合物可採用各種微脂粒形式,諸如脂質體、微胞或乳液。陽離子性脂質複合物可為單層或多層複合物。當包括抗原時,該抗原可封裝於陽離子性脂質複合物內或可不封裝。應瞭解封裝意謂抗原可包含於複合物之內部空間內且/或併入複合物之脂質壁內。
本發明進一步係關於一種製備該等複合物的方法,其中該方法視需要可包括將該等調配物自個別過量組分中純化的步驟。對於本發明之抗原複合物之製備,有利實施例包
含純化步驟。
在某些實施例中,陽離子性脂質複合物在pH 6.0-8.0時具有淨正電荷及/或帶正電荷之表面。
可與本發明之陽離子性脂質複合物一起包括在內的可選"抗原"可為核酸、肽、脂肽、蛋白質、脂蛋白、多醣及可與陽離子性脂質直接複合之其他巨分子。然而,陽離子性藥物(例如大的陽離子蛋白質)可與陰離子性脂質直接複合,或依序地,首先與陰離子性脂質或聚合物複合、繼之與陽離子性脂質複合。使用該方法容許藉由本發明之複合物將帶正電或不帶電藥物傳遞至細胞中。
本發明之一態樣涉及使用陽離子性脂質複合物刺激趨化因子及細胞因子之產生。趨化因子及細胞因子為免疫反應之重要調節劑。趨化因子最初鑑定為炎性細胞(包括嗜中性白細胞、嗜伊紅細胞及單核細胞/巨噬細胞)的強化學引誘劑(chemoattractant)。隨後研究揭露,趨化因子藉由調節樹突狀細胞及其他淋巴細胞進入淋巴器官內之通行而對免疫反應具有深遠影響。樹突狀細胞為在組織中選取抗原、遷移至引流淋巴結並成熟以刺激T細胞反應的游走細胞。CC趨化因子中之一成員CCL2最初鑑定為單核細胞/巨噬細胞之趨化及活化因子。隨後研究表明,其亦可影響T細胞、天然殺手細胞及嗜中性白細胞之功能。深入調查發現,當存在Th1細胞因子、介白素-12("IL-12")及干擾素-γ("IFN-γ")時,CCL2為CD8+
細胞毒性T淋巴細胞("CTL")活性之最強活化劑。此可藉由CCL2與IFN-γ系統之間的雙向
正相互作用來解釋。細胞因子或趨化因子之缺乏會干擾Th1極化及隨後的特異性腫瘤免疫形成。另一種CC趨化因子CCL-4亦已表明可活體內募集並擴增樹突狀細胞並增強質體DNA疫苗之免疫原性。最近已證明,趨化因子藉由將純真(naive)CD8+
T細胞導引至CD4+
T細胞-樹突狀細胞相互作用之部位來增強免疫性並促進記憶CD8+
T細胞形成。可藉由本發明之陽離子性脂質複合物刺激之趨化因子之一些實例為CCL-2、CCL-3及CCL-4。可藉由本發明之陽離子性脂質複合物刺激之細胞因子之實例為IL-12及IFN-γ。本發明人預期,本發明之陽離子性脂質複合物亦可刺激除本說明書中所揭示之趨化因子及細胞因子外的趨化因子及細胞因子。
在另一態樣中,本發明之陽離子性脂質複合物藉由活化細胞激酶途徑(諸如細胞外信號調節激酶(ERK)途徑(亦名有絲分裂原活化(MAP)激酶途徑)、p38途徑或磷脂醯肌醇-3(PI-3)途徑)來刺激免疫反應。該等途徑又可對刺激免疫反應及產生細胞因子及趨化因子加以調節。該等途徑已為熟習此項技術者所熟知。
本發明之陽離子性脂質複合物可調節T細胞活性以刺激免疫反應。T細胞有三類:輔助T細胞、殺手T細胞及調節性T細胞。該等三類T細胞合作協調細胞免疫反應。細胞毒性T-淋巴細胞("CTL")(亦名殺手T細胞或CD8+
T-細胞)負責攻擊表現外來或腫瘤抗原之細胞。咸信調節性T細胞負責減弱CTL介導之免疫性,但調節性T細胞之此作用的確切
機制尚不瞭解。已知降低調節性T細胞活性可形成更強CTL活性,引起更強細胞免疫反應。如下文實例中所示,本發明之陽離子性脂質複合物在最佳脂質劑量及組成下亦可藉由減少調節性T細胞群來刺激強免疫反應。
本發明之陽離子性脂質複合物可形成視需要與抗原混合的脂質體且可僅含陽離子性脂質或含有陽離子性脂質與中性脂質之組合。適當的陽離子性脂質物質包括但不限於:
3-β[4
N-(1
N,8
-二胍基亞精胺)-胺甲醯基]膽固醇(BGSC);3-β[N,N-二脈基乙基-胺基乙烷)-胺甲醯基]膽固醇(BGTC);N,N1
N2
N3
四甲基四棕櫚醯基精胺(Cellfectin);N-第三丁基-N'-十四烷基-3-十四烷基-胺基丙脒(CLONfectin);溴化二甲基二-十八烷基銨(DDAB);溴化1,2-二-十四烷基氧基丙基-3-二甲基-羥基乙基銨(DMRIE);2,3-二油醯基氧基-N-[2(精胺甲醯胺基)乙基]-N,N-二甲基-1-丙烷三氟乙酸銨(DOSPA);1,3-二油醯基氧基-2-(6-carboxyspermyl)-丙基醯胺(DOSPER);4-(2,3-雙-棕櫚醯基氧基-丙基)-1-甲基-1H-咪唑(DPIM);碘化N,N,N',N'-四甲基-N,N'-雙(2-羥基乙基)-2,3-二油醯基氧基-1,4-丁烷二銨(Tfx-50);氯化N-1-(2,3-二油醯基氧基)丙基-N,N,N-三甲基銨(DOTMA)或其他N-(N,N-1-二烷氧基)-烷基-N,N,N-三取代銨界面活性劑;1,2二油醯基-3-(4'-三甲基銨基)丁醇-sn-甘油(DOBT)或膽固醇基(4'三甲基銨)丁酸鹽(ChOTB),其中三甲基銨基團經由丁醇間隔臂連接至雙
鏈(對於DOTB)或膽固醇基(對於ChOTB);如WO 93/03709中所揭示的DORI(DL-1,2-二油醯基-3-二甲基胺基丙基-β-羥基乙基銨)或DORIE(DL-1,2-O-二油醯基-3-二甲基胺基丙基-β-羥基乙基銨)(DORIE)或其類似物;1,2-二油醯基-3-琥珀醯基-sn-甘油膽鹼酯(DOSC);膽固醇基半琥珀酸酯(ChOSC);脂多胺,諸如二-十八烷基醯胺基甘胺醯基精胺(DOGS)及二棕櫚醯基磷脂醯基乙醇戊基精胺(DPPES)或如美國專利第5,283,185號中所揭示的陽離子性脂質;碘化膽固醇基-3β-羧基-醯胺基-伸乙基三甲基銨、碘化1-二甲基胺基-3-三甲基銨基-DL-2-丙基-膽固醇基甲酸酯、膽固醇基-3-O-甲醯胺基伸乙胺、碘化膽固醇基-3-β-氧基琥珀醯胺基-伸乙基三甲基銨、碘化1-二甲基胺基-3-三甲基銨基-DL-2-丙基-膽固醇基-3-β-氧基琥珀酸酯、碘化2-(2-三甲基銨基)-乙基甲基胺基乙基-膽固醇基-3-β-氧基琥珀酸酯、3-β-N-(N',N'-二甲基胺基乙烷)胺甲醯基膽固醇(DC-chol),及3-β-N-(聚伸乙亞胺)-胺甲醯基膽固醇;O,O'-二-十四烷基-N-離胺醯基天冬胺酸酯(DMKE);O,O'-二-十四烷基-N-離胺醯基-麩胺酸酯(DMKD);溴化1,2-二-十四烷基氧基丙基-3-二甲基-羥基乙基銨(DMRIE);1,2-二月桂醯基-sn-甘油基-3-乙基磷酸膽鹼(DLEPC);1,2-二肉豆蔻醯基-sn-甘油基-3-乙基磷酸膽鹼(DMEPC);1,2-二油醯基-sn-甘油基-3-乙基磷酸膽鹼(DOEPC);1,2-二棕櫚醯基-sn-甘油基-3-乙基磷酸膽鹼(DPEPC);1,2-二硬脂醯基-sn-甘油基-3-乙基磷酸膽鹼(DSEPC);1,2-二油醯基-3-三甲基銨丙烷
(DOTAP);二油醯基二甲基胺基丙烷(DODAP);1,2-棕櫚醯基-3-三甲基銨丙烷(DPTAP);1,2-二硬脂醯基-3-三甲基銨丙烷(DSTAP)、1,2-肉豆蔻醯基-3-三甲基銨丙烷(DMTAP);及十二烷基硫酸鈉(SDS)。本發明涵蓋使用本申請案中所揭示之陽離子性脂質之結構變體及衍生物。
本發明之某些態樣包括具有由以下式所示之結構的非類固醇陽離子性脂質:
其中R1
為四級銨基團,Y1
係選自烴鏈、酯、酮及肽,R2
及R3
獨立地選自飽和脂肪酸、不飽和脂肪酸、酯連接之烴、磷酸二酯及其組合。DOTAP、DMTAP、DSTAP、DPTAP、DPEPC、DSEPC、DMEPC、DLEPC、DOEPC、DMKE、DMKD、DOSPA、DOTMA為具有該一般結構之脂質實例。
在一實施例中,本發明之陽離子性脂質為其中親脂性基團與胺基之間的鍵在水溶液中穩定的脂質。因此,本發明之複合物之特性為其在儲存期間的穩定性(亦即,其能夠在形成後隨時間維持小直徑且保持生物活性)。陽離子性脂質中所用的該等鍵包括醯胺鍵、酯鍵、醚鍵及胺甲醯鍵。熟習此項技術者易瞭解,可使用含有一種以上陽離子性脂質物質的脂質體製備本發明之複合物。舉例而言,已揭示供某些藥物傳遞應用之包含離胺醯基-磷脂醯乙醇胺
及β-丙胺醯基膽固醇酯兩種陽離子性脂質物質的脂質體[Brunette, E.等人,Nucl. Acids Res., 20:1151 (1992)]。
此外應瞭解,在考量適用於本發明且視需要與抗原混合之陽離子性脂質體時,本發明之方法並非僅限於使用上述陽離子性脂質,而是可使用任何脂質組合物,只要可製備陽離子性脂質體且所得陽離子電荷密度足以活化並誘導免疫反應即可。
因此,除陽離子性脂質外,本發明之複合物亦可含有其他脂質。該等脂質包括但不限於:溶血脂質,例如溶血磷脂醯膽鹼(1-油醯基溶血磷脂醯膽鹼);膽固醇;或中性磷脂,包括二油醯基磷脂醯乙醇胺(DOPE)或二油醯基磷脂醯膽鹼(DOPC);以及含有聚乙二醇部分的各種親脂性界面活性劑,例如Tween-80及PEG-PE。
本發明之陽離子性脂質複合物亦可含有帶負電荷的脂質以及陽離子性脂質,只要所形成之複合物之淨電荷為正且/或複合物之表面帶正電荷即可。本發明之帶負電荷脂質為包含至少一種在生理性pH值下或接近生理性pH值時具有淨負電荷之脂質物質或該等脂質物質之組合的彼等脂質。帶負電荷的適當脂質物質包括但不限於CHEMS(膽固醇基半琥珀酸酯)、NGPE(N-戊二醯基磷脂醯乙醇胺)、磷脂醯甘油及磷脂酸或相似的磷脂類似物。
用於製備本發明之包含脂質之藥物傳遞複合物之脂質體的製備方法已為一般熟習此項技術者所知。脂質體製備方法之評述可見於Liposome Technology (CFC Press New
York 1984);Ostro之Liposomes (Marcel Dekker, 1987);Methods Biochem Anal. 33:337-462 (1988)及美國專利第5,283,185號。該等方法包括凍融擠出法及超音波處理法。單層脂質體(平均直徑小於約200 nm)與多層脂質體(平均直徑大於約300 nm)可用作製備本發明之複合物的起始組分。
在用於製備本發明之陽離子性脂質複合物的陽離子性脂質體中,陽離子性脂質以全部脂質體脂質之約10莫耳%至約100莫耳%或約20莫耳%至約80莫耳%存在於脂質體中。
中性脂質當包括於脂質體內時,可以全部脂質體脂質之約0莫耳%至約90莫耳%或約20莫耳%至約80莫耳%或40莫耳%至80莫耳%之濃度存在。帶負電荷的脂質當包括於脂質體內時可以全部脂質體脂質之約0莫耳%至約49莫耳%或約0莫耳%至約40莫耳%範圍內之濃度存在。在一實施例中,脂質體含有比率介於約2:8至約6:4之間的陽離子性脂質與中性脂質。此外應瞭解,本發明之複合物可含有經修飾之脂質、蛋白質、聚陽離子或受體配位體,起靶向因子之作用,從而將複合物引導至特定組織或細胞類型。靶向因子之實例包括但不限於去唾液酸醣蛋白質(asialoglycoprotein)、胰島素、低密度脂蛋白(LDL)、葉酸鹽及針對細胞表面分子之單株及多株抗體。此外,為修改複合物之循環半衰期,可藉由合併含有聚乙二醇部分的親脂性界面活性劑來將表面正電荷加以空間屏蔽。
陽離子性脂質複合物經由蔗糖梯度收集後可儲存於等張
性蔗糖或右旋糖溶液中,或可將其凍乾且隨後在使用之前於等張溶液中復水。在一實施例中,陽離子性脂質複合物儲存於溶液中。本發明之陽離子性脂質複合物之穩定性可藉由特定檢定量測,以測定陽離子性脂質複合物在儲存期間隨時間的物理穩定性及生物活性。陽離子性脂質複合物之物理穩定性可藉由一般熟習此項技術者已知之方法(包括例如電子顯微術、凝膠過濾層析或藉助於準彈性光散射)、使用如實例中所述的例如Coulter N4SD粒徑分析儀測定陽離子性脂質複合物之直徑及電荷加以量測。當所儲存之陽離子性脂質複合物之直徑與在陽離子性脂質複合物純化時所測定之陽離子性脂質複合物直徑相比,增加不超過100%或不超過50%或不超過30%時,稱陽離子性脂質複合物之物理穩定性在儲存期間"實質不變"。
儘管陽離子性脂質可以純形式或實質純形式投藥,但較佳將其作為醫藥組合物、調配物或製劑提供。使用本發明之陽離子性脂質複合物的醫藥調配物可包含處於生理相容性無菌緩衝液中的陽離子性脂質複合物,該緩衝液諸如磷酸鹽緩衝鹽水、等張鹽水或低離子濃度緩衝液,諸如乙酸鹽或Hepes(例示性pH值在約5.0至約8.0範圍內)。陽離子性脂質複合物可作為供瘤內、動脈內、靜脈內、氣管內、腹膜內、皮下及肌內投藥之氣霧劑或液體溶液投藥。
本發明之調配物可合併此項技術中已知的任何穩定劑。說明性穩定劑為膽固醇及可有助於固定脂質體雙層並防止雙層解體或失穩的其他固醇。此外,可將諸如聚乙二醇、
多醣及單醣之藥劑併入脂質體內,以修飾脂質體表面並防止其因與血液組分相互作用而失穩。其他說明性穩定劑為可單獨使用或混合使用的蛋白質、醣類、無機酸或有機酸。
可使用多種藥學方法控制、修改或延長免疫刺激之持續時間。控制釋放型製劑可經由使用封裝或捕集陽離子性脂質並將其緩慢釋放的聚合物複合物(諸如聚酯、聚胺基酸、甲基纖維素、聚乙烯、聚(乳酸)及水凝膠)獲得。亦可使用類似的聚合物吸附脂質體。脂質體可包含於乳液調配物中,以便改變刺激劑之釋放分布。或者,刺激劑存在於血液循環中的持續時間可藉由用諸如聚乙二醇或其他聚合物及其他物質(諸如能夠延長脂質體及乳液之循環時間或半衰期的醣類)之化合物塗佈脂質體之表面來延長。
當需要口服製劑時,可將陽離子性脂質與此項技術中已知的一般醫藥載劑組合,該等醫藥載劑諸如蔗糖、乳糖、甲基纖維素、羧甲基纖維素或阿拉伯膠(gum Arabic)及其他載劑。陽離子性脂質亦可封裝於膠囊或錠劑中供全身性傳遞。
本發明之陽離子性脂質可針對預防性或治療性目的投藥。當預防性給藥時,陽離子性脂質可先於疾病之任何跡象或症狀提供。當治療性給藥時,陽離子性脂質在疾病發作時或發作之後提供。免疫刺激劑之治療性投藥可供減輕或治癒疾病之用。為一舉兩得,可將陽離子性脂質與另一種治療劑或抗原一起投藥。當將陽離子性脂質與另一種治
療劑或抗原一起投藥時,可產生針對特定疾病的預防性或治療性作用。
為獸醫學與人類用途起見,本發明之調配物包含如上所述之單獨陽離子性脂質,以及視需要一或多種治療性成分,諸如抗原或藥物分子。調配物可方便地以單位劑型提供且可藉由醫藥技術中已知之任何方法製備。
在一實施例中,陽離子性脂質在其他任何藥劑不存在下投藥,以便激發或降低多種免疫反應(包括產生其他免疫調節劑)及激發免疫反應對抗疾病。在另一實施例中,將陽離子性脂質與一或多種抗原組合投藥。在此情況下,旨在產生特異於與陽離子性脂質組合傳遞之抗原的免疫反應。所產生的反應可包括產生特異性細胞毒性T-細胞、記憶T-細胞或B-細胞,從而預防與彼等抗原相關的特定疾病或形成針對與彼等抗原相關之特定疾病的治療性反應。抗原可為任何腫瘤相關抗原或微生物抗原或熟習此項技術者已知的其他任何抗原。
在MHC分子之情形下,如本文中所使用的"腫瘤相關抗原"為與腫瘤或癌細胞相關且當在抗原呈現細胞之表面上受表現時能夠激發免疫反應(體液及/或細胞免疫反應)的分子或化合物(例如蛋白質、肽、多肽、脂蛋白、脂肽、醣蛋白、醣肽、脂質、醣脂、碳水化合物、RNA及/或DNA)。腫瘤相關抗原包括自體抗原,以及可能並非與癌症特定相關、但投與動物時仍然增強針對腫瘤或癌細胞之
免疫反應及/或減緩腫瘤或癌細胞生長的其他抗原。本文中提供更多特定實施例。
如本文中所使用的"微生物抗原"為微生物之抗原且包括但不限於感染性病毒、感染性細菌、感染性寄生蟲及感染性真菌。微生物抗原可為完整微生物及其天然分離株、片段或衍生物、與天然存在之微生物抗原相同或類似且較佳誘導特異於相應微生物(天然存在之微生物抗原所來源之微生物)之免疫反應的合成化合物。在一較佳實施例中,若一種化合物誘導之免疫反應(體液及/或細胞免疫反應)類似於天然存在之微生物抗原,則該化合物類似於天然存在之微生物抗原。一般熟習此項技術者熟知類似於天然存在之微生物抗原的化合物或抗原,諸如蛋白質、肽、多肽、脂蛋白、脂肽、醣蛋白、醣肽、脂質、醣脂、碳水化合物、RNA及/或DNA。與天然存在之微生物抗原類似之化合物之另一非限定實例為多醣抗原之肽模擬物。本文中提供更多的特定實施例。
術語"抗原"進一步旨在涵蓋已知抗原或野生型抗原之肽或蛋白質類似物,諸如本說明書中所述的彼等物。該等類似物可能比野生型抗原更易溶或更穩定,且亦可能含有賦予抗原更強免疫活性的突變或修飾。抗原可以任何方式修飾,諸如添加脂質或糖部分、使肽或蛋白質胺基酸序列突變、使DNA或RNA序列突變或熟習此項技術者已知的其他任何修飾。抗原可使用熟習此項技術者已知的標準方法加以修飾。
具有與所要抗原之胺基酸序列同源之胺基酸序列的肽或蛋白質亦適用於本發明之組合物及方法,其中同源抗原誘導針對相應腫瘤、微生物或受感染細胞的免疫反應。
在一實施例中,陽離子性脂質複合物中之抗原包含與腫瘤或癌症相關的抗原,亦即腫瘤相關抗原,以產生預防或治療腫瘤之疫苗。因而,在一實施例中,本發明之腫瘤或癌症疫苗進一步包含至少一種腫瘤相關抗原之至少一抗原決定基。在另一較佳實施例中,本發明之腫瘤或癌症疫苗進一步包含來源於一或多種腫瘤相關抗原的複數個抗原決定基。在本發明之陽離子性脂質複合物及方法中獲得應用的腫瘤相關抗原可具有固有免疫原性或非免疫原性或微弱免疫原性。如本文中所證明,即使腫瘤相關性自體抗原亦可有利地用於本發明疫苗中達成治療性作用,因為本發明組合物能夠破壞針對該等抗原的免疫耐受性。例示性抗原包括但不限於合成抗原、重組抗原、外來抗原或同源抗原,且抗原物質可包括但不限於蛋白質、肽、多肽、脂蛋白、脂肽、脂質、醣脂、碳水化合物、RNA及DNA。該等疫苗之實例包括但不限於使用與腫瘤相關抗原複合的陽離子性脂質治療或預防乳癌、頭頸癌、黑色素瘤、子宮頸癌、肺癌、前列腺癌、腸癌,或此項技術中已知之可經受免疫治療的其他任何癌症。亦可將抗原與陽離子性脂質一起調配,而不封裝於脂質體中。
本發明之陽離子性脂質複合物可用於癌症治療或預防方法中。在此情況下,對待免疫的哺乳動物注射含有具有封
裝抗原之脂質體的醫藥調配物。可用腫瘤疫苗治療之癌症之實例包括但不限於使用陽離子性脂質及與癌症相關的抗原或多種肽抗原預防或治療乳癌、頭頸癌、黑色素瘤、子宮頸癌、肺癌、前列腺癌、腸癌,或此項技術中已知的其他任何癌症。亦可將抗原與陽離子性脂質一起調配而不將抗原封裝於脂質體中。
適用於本發明的腫瘤相關抗原包括天然存在之分子與經修飾之分子,其可在幾種腫瘤類型中指示單一腫瘤類型,且/或與正常細胞相比,僅在腫瘤細胞中受到表現或過度表現。除蛋白質、醣蛋白、脂蛋白、肽及脂肽外,碳水化合物、神經節苷脂、醣脂及黏蛋白(mucin)之表現之腫瘤特異性模式亦已得到證明。用於癌症疫苗的例示性腫瘤相關抗原包括致癌基因、腫瘤抑制基因及具有腫瘤細胞所特有之突變或重排之其他基因的蛋白質產物;再活化胚基因產物;癌胚抗原;組織特異性(而非腫瘤特異性)分化抗原;生長因子受體;細胞表面碳水化合物殘基;外來病毒蛋白質;及其他多種自體蛋白質。
腫瘤相關抗原之特定實施例包括:例如,突變或修飾抗原,諸如Ras p21原癌基因、腫瘤抑制因子p53及HER-2/neu及BCR-Abl致癌基因之蛋白質產物,以及CDK4、MUM1、卡斯蛋白酶8(Caspase 8)及β索烴素(Beta catenin);過度表現之抗原,諸如半乳糖凝集素4(galectin 4)、半乳糖凝集素9、碳酸酐酶、醛縮酶A(Aldolase A)、PRAME、Her2/neu、ErbB-2及KSA;癌胚抗原,諸如α胎
蛋白(alpha fetoprotein;AFP)、人類絨毛膜促性腺激素(hCG);自體抗原,諸如癌胚抗原(CEA)及黑色素細胞分化抗原,諸如Mart 1/Melan A、gp100、gp75、酪胺酸酶、TRP1及TRP2;前列腺相關抗原,諸如PSA、PAP、PSMA、PSM-P1及PSM-P2;再活化胚基因產物,諸如MAGE1、MAGE3、MAGE4、GAGE1、GAGE2、BAGE、RAGE,及其他癌睾丸抗原,諸如NY-ESO1、SSX2及SCP1;黏蛋白,諸如Muc-1及Muc-2;神經節苷脂,諸如GM2、GD2及GD3;中性醣脂及醣蛋白,諸如Lewis(y)及globo-H;及醣蛋白,諸如Tn、Thompson-Freidenreich抗原(TF)及sTn。本文中的腫瘤相關抗原亦包括完整細胞及腫瘤細胞溶胞液,以及其免疫原性部分,以及在單株增殖之B淋巴細胞上受到表現、針對B細胞淋巴瘤使用的免疫球蛋白個體基因型。
如癌瘤之抗原,腫瘤相關抗原及其相應的腫瘤靶細胞包括例如細胞角蛋白(cytokeratin),尤其細胞角蛋白8、細胞角蛋白18及細胞角蛋白19。上皮膜抗原(EMA)、人類胚抗原(HEA-125)、人乳脂肪球、MBr1、MBr8、Ber-EP4、17-1A、C26及T16亦為已知的癌瘤抗原。結蛋白(Desmin)及肌肉特異性肌動蛋白(actin)為肌原性肉瘤之抗原。胎盤鹼性磷酸酶、β-人類絨毛膜促性腺激素及α胎蛋白為滋養層及生殖細胞腫瘤之抗原。前列腺特異性抗原為前列腺癌之抗原、結腸腺癌之癌胚抗原。HMB-45為黑色素瘤之抗原。
在子宮頸癌中,適用抗原可由人類乳頭狀瘤病毒編碼。嗜
鉻粒蛋白-A(Chromagranin-A)及突觸素(synaptophysin)為神經內分泌及神經外胚層腫瘤之抗原。特別受關注的是侵入性腫瘤,其形成具有壞死區之實體腫瘤塊。該等壞死細胞之溶解為抗原呈現細胞之抗原富集源,且因此本發明之療法可有利地結合習知化學療法及/或放射療法使用。
在一實施例中,使用人類乳頭狀瘤病毒HPV抗原。用作腫瘤相關抗原之特異性HPV抗原為HPV亞型16 E7。HPV E7抗原-陽離子性脂質複合物可有效預防及治療子宮頸癌。此外,具有抗原活性、但無致瘤活性之遺傳工程E7蛋白質(亦即E7m蛋白質)為有效的腫瘤相關抗原。E7m-陽離子性脂質複合物誘導細胞免疫性以促成所形成之腫瘤完全消退,且因此可用作強抗子宮頸癌疫苗。
腫瘤相關抗原可藉由此項技術中熟知的方法製備。舉例而言,該等抗原可藉由製備癌細胞之粗萃取物(例如,如Cohen等人,Cancer Res., 54:1055 (1994)中所述)、藉由部分地純化抗原、藉由重組技術或藉由已知抗原之重新合成,由癌細胞製備。抗原亦可為編碼抗原肽之核酸形式,該抗原肽為適於在受檢者中受表現且適於呈現給所免疫受檢者之免疫系統的形式。此外,抗原可為全抗原,或其可為包含至少一抗原決定基之全抗原的片段。
本發明之癌症疫苗中亦可有利地包括已知易感染為某些癌症之病原體所衍生的抗原。據估計,全世界癌症發病率中接近16%可歸因於感染性病原體;且多種一般惡性腫瘤的特徵為表現特定病毒基因產物。因此,包合一或多種來
源於涉及引起癌症之病原體的抗原可有助於擴大宿主免疫反應且增強癌症疫苗之預防性或治療性作用。特受關注用於本文中所提供之癌症疫苗中的病原體包括:B型肝炎病毒(肝細胞癌)、C型肝炎病毒(肝腫瘤)、艾普斯坦-巴爾病毒(Epstein Barr virus,EBV)(在免疫抑制個體中為伯基特氏(Burkitt)淋巴瘤、鼻咽癌、PTLD)、HTLVL(成人T細胞白血病)、致癌性人類乳頭狀瘤病毒16、18、33、45型(成人子宮頸癌),及細菌幽門螺旋桿菌(B細胞胃淋巴瘤)。在哺乳動物及更尤其人類中可用作抗原的其他醫學相關微生物廣泛描述於文獻中,例如C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983,其整個內容以引用方式併入本文中。
在另一實施例中,陽離子性脂質複合物中之抗原包含衍生於病原體或與病原體相關的抗原,亦即微生物抗原。因而,在一實施例中,本發明之病原體疫苗進一步包含至少一種微生物抗原之至少一抗原決定基。可被本發明疫苗靶向的病原體包括但不限於病毒、細菌、寄生蟲及真菌。在另一實施例中,本發明之病原體疫苗進一步包含來源於一或多種微生物抗原的複數個抗原決定基。在該等陽離子性脂質複合物及方法中獲得應用的微生物抗原可具有固有免疫原性或非免疫原性或微弱免疫原性。例示性抗原包括但不限於合成抗原、重組抗原、外來抗原或同源抗原,且抗原物質可包括但不限於蛋白質、肽、多肽、脂蛋白、脂肽、脂質、醣脂、碳水化合物、RNA及DNA。
例示性病毒性病原體包括但不限於感染哺乳動物及更尤其人類的病毒。病毒之實例包括但不限於:逆轉錄病毒科(Retroviridae)(例如人類免疫缺乏病毒,諸如HIV-1(亦稱為HTLV-III、LAV或HTLV-III/LAV,或HIV-III;及其他分離株,諸如HIV-LP);小核醣核酸病毒科(Picornaviridae)(例如小兒麻痹病毒(polio viruses)、A型肝炎病毒;腸病毒、人類柯沙奇病毒(human Coxsackie virus)、鼻病毒(rhinoviruses)、伊科病毒(echo viruses));杯狀病毒科(Calciviridae)(例如導致胃腸炎的病毒株);披膜病毒科(Togaviridae)(例如馬腦炎病毒、風疹病毒);黃病毒科(Flaviridae)(例如登革熱病毒(dengue virus)、腦炎病毒、黃熱病病毒);冠狀病毒科(Coronoviridae)(例如冠狀病毒);彈狀病毒科(Rhabdoviradae)(例如水泡性口炎病毒、狂犬病病毒);冠狀病毒科(例如冠狀病毒);彈狀病毒科(例如水泡性口炎病毒、狂犬病病毒);線病毒科(Filoviridae)(例如伊波拉病毒(ebola virus));副黏病毒科(Paramyxoviridae)(例如副流感病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞體病毒);正黏液病毒科(Orthomyxoviridae)(例如流感病毒);布尼亞病毒科(Bungaviridae)(例如漢坦病毒(Hantaan virus)、班伽病毒(bunga virus)、沙蠅病毒(phlebovirus)及奈洛病毒(Nairo virus));沙狀病毒科(Arena viridae)(出血熱病毒);裏奧病毒科(Reoviridae)(例如裏奧病毒(reoviruses)、環狀病毒(orbiviurses)及輪狀病毒(rotaviruses));雙核糖核酸病毒科(Birnaviridae);肝炎病
毒科(Hepadnaviridae)(B型肝炎病毒);細小病毒科(Parvovirida)(細小病毒);乳多空病毒科(Papovaviridae)(乳頭狀瘤病毒、多形瘤病毒);腺病毒科(Adenoviridae)(大部分為腺病毒);疱疹病毒科(Herpesviridae)(單純性疱疹病毒(HSV)1及2、水痘帶狀疱疹病毒(varicella zoster virus)、巨細胞病毒(CMV)、疱疹病毒);痘病毒科(Poxyiridae)(天花病毒(variola viruses)、牛痘病毒(vaccinia viruses)、痘病毒(pox viruses));及虹彩病毒科(Iridoviridae)(例如非洲豬瘟病毒(African swine fever virus));及未分類病毒(例如海綿狀腦病變之病原體、delta 型肝炎病原體(認為是B型肝炎病毒之缺失性衛星(defective satellite))、非A型肝炎、非B型肝炎之病原體(1類=內部傳播;2類=非經腸傳播(亦即C型肝炎);諾瓦克(Norwalk)及相關病毒,及星狀病毒(astroviruses))。
此外,在脊椎動物中,本發明組合物及方法可靶向革蘭氏陰性(gram negative)及革蘭氏陽性細菌。該等革蘭氏陽性細菌包括但不限於巴氏桿菌屬種(Pasteurella
species)、葡萄球菌屬種(Staphylococci
species)及鏈球菌屬種(Streptococcus
species)。革蘭氏陰性細菌包括但不限於大腸埃希氏桿菌(Escherichia
coli)、假單胞菌屬種(Pseudomonas
species)及沙門氏菌屬種(Salmonella
species)。感染性細菌之特定實例包括但不限於:幽門螺桿菌(Helicobacter pyloris
)、柔氏螺旋體菌(Borella burgdorferi
)、嗜肺性退伍軍人桿菌(Legionella pneumophilia
)、分枝桿菌屬種
(Mycobacteria
sps)(例如結核分枝桿菌(M. tuberculosis
)、鳥分枝桿菌(M. avium
)、細胞內分枝桿菌(M. intracellular
)、坎沙西分枝桿菌(M. kansaii
)、戈登分支桿菌(M. gordonae
))、金黃色葡萄球菌(Staphylococcus aureus
)、淋病奈瑟氏球菌(Neisseria gonorrhoeae
)、腦膜炎奈瑟氏菌(Neisseria meningitidis
)、單核細胞增生利斯特氏菌(Listeria monocytogenes
)、化膿性鏈球菌(Streptococcus pyogenes
)(A群鏈球菌)、無乳鏈球菌(Streptococcus agalactiae
)(B群鏈球菌)、鏈球菌屬(草綠色鏈球菌群(viridans
group))、糞鏈球菌(Streptococcus faecalis
)、牛鏈球菌(Streptococcus bovis
)、鏈球菌屬(厭氧菌種)、肺炎鏈球菌(Streptococcus pneumoniae
)、病原性彎曲桿菌屬種(pathogenicCampylobacter
sp.)、腸球菌種(Enterococcus
sp.)、流感嗜血桿菌(Haemophilus infuenzae
)、炭疽桿菌(Bacillus antracis
)、白喉棒狀桿菌(corynebacterium diphtheriae
)、棒狀桿菌屬種(corynebacterium
sp.)、紅斑丹毒絲菌(Erysipelothrix rhusiopathiae
)、產氣莢膜梭茵(Clostridium perfringers
)、破傷風梭菌(Clostridium tetani
)、產氣腸桿菌(Enterobacter aerogenes
)、肺炎克雷伯氏桿菌(Klebsiella pneumoniae
)、敗血性巴氏桿菌(Pasturella multocida
)、擬桿菌種(Bacteroides
sp.)、具核梭桿菌(Fusobacterium nucleatum
)、念珠狀鏈桿菌(Streptobacillus moniliformis
)、梅毒螺旋體菌(Treponema pallidium
)、細弱密螺旋體菌(Treponema pertenue
)、鉤端螺旋體屬(Leptospira
)、立克次氏體屬
(Rickettsia
)及伊氏放線菌(Actinomyces israelii
)。
可作為微生物抗原之來源用於本發明組合物中的細菌性病原體之多肽包括但不限於:導致癤病(furunculosis)之殺鮭氣單胞菌(Aeromonis salmonicida
)之鐵調節外膜蛋白("IROMP")、外膜蛋白("OMP")及A蛋白,導致細菌性腎病("BKD")之鮭魚腎桿菌(Renibacterium salmoninarum
)之p57蛋白,耶爾森氏菌病(Yersiniosis
)之主要表面結合抗原("msa")、表面表現型細胞毒素("mpr")、表面表現型溶血素("ish")及鞭毛型抗原;巴斯德氏菌病(Pasteurellosis
)之細胞外蛋白("ECP")、鐵調節型外膜蛋白("IROMP")及結構蛋白;鰻弧菌病(Vibrosis anguillarum
)及奧德弧菌(V. ordalii
)之OMP及鞭毛蛋白;鯰魚愛德華氏菌病(Edwardsiellosis ictaluri
)及遲鈍愛德華氏菌病(E. tarda
)之鞭毛蛋白、OMP蛋白、aroA及purA;及小瓜蟲病(Ichthyophthirius
)之表面抗原;及柱狀噬纖維菌病(Cytophaga columnari
)之結構及調節蛋白;及立克次體(Rickettsia
)之結構及調節蛋白。該等抗原可藉由重組或藉由此項技術中已知的其他任何方式分離或製備。
病原體之實例進一步包括但不限於感染哺乳動物及更尤其人類的真菌。真菌之實例包括但不限於:新型隱球菌(Cryptococcus neoformans
)、莢膜組織胞漿菌(Histoplasma capsulatum
)、粗球孢子菌(Coccidioides immitis
)、皮炎芽生菌(Blastomyces dermatitidis
)、砂眼披衣菌(Chlamydia trachomatis
)、白色念珠菌(Candida albicans
)。感染性寄
生蟲之實例包括瘧原蟲(Plasmodium
),諸如鐮狀瘧原蟲(Plasmodium falciparum
)、三日瘧原蟲(Plasmodium malariae
)、卵形瘧原蟲(Plasmodium ovale
)及間日瘧原蟲(Plasmodium vivax
)。其他感染性有機體(亦即原生生物(protist))包括剛地弓形蟲(Toxoplasma gondii
)。寄生性病原體之多肽包括但不限於小瓜蟲病之表面抗原。
在哺乳動物及更尤其人類中可用作抗原的其他醫學相關微生物廣泛描述於文獻中,例如參見C. G. A Tbomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983,其整個內容以引用方式併入本文中。除治療感染性人類疾病及人類病原體外,本發明之組合物及方法亦可用於治療非人類哺乳動物之感染。供治療非人類哺乳動物之用的多種疫苗揭示於Bennett, K. Compendium of Veterinary Products,第3版,North American Compendiums, Inc., 1995;亦參見WO 02/069369,該等文獻之揭示內容以引用方式明確併入本文中。
例示性非人類病原體包括但不限於:小鼠乳腺瘤病毒(mouse mammary tumor virus,"MMTV")、勞斯肉瘤病毒(Rous sarcoma virus,"RSV")、禽白血病病毒(avian leukemia virus,"ALV")、禽骨髓母細胞瘤病毒(avian myeloblastosis virus,"AMV"))、鼠白血病病毒(murine leukemia virus,"MLV")、貓白血病毒(feline leukemia virus,"FeLV")、鼠肉瘤病毒(murine sarcoma virus,"MSV")、長臂猿白血病病毒(gibbon ape leukemia virus,
"GALV")、脾壞死病毒(spleen necrosis virus,"SNV")、網狀內皮增殖病病毒(reticuloendotheliosis virus,"RV")、猿猴肉瘤病毒(simian sarcoma virus,"SSV")、Mason-Pfizer猴病毒(Mason-Pfizer monkey virus,"MPMV")、猿猴1型逆轉錄病毒(simian retrovirus type 1,"SRV-1")、慢病毒(lentiviruses)(諸如HIV-1、HIV-2、SIV、脫髓鞘性腦白質炎的病毒(Visna virus)、貓免疫缺乏病毒(feline immunodeficiency virus,"FIV")及馬傳染性貧血病毒(equine infectious anemia virus,"EIAV"))、T-細胞白血病病毒(諸如HTLV-1、HTLV-II、猿猴T-細胞白血病病毒(simian T-cell leukemia virus,"STLV")及牛白血病病毒(bovine leukemia virus,"BLV")),及泡沫病毒(foamy viruses),諸如人類泡沫病毒(human foamy virus,"HFV")、猿猴泡沫病毒(simian foamy virus,"SFV")及牛泡沫病毒(bovine foamy virus,"BFV")。
在有些實施例中,如本文中提及感染性病原體時所使用的"治療"係指增強受檢者抗病原體感染之能力或使受檢者感染上病原體之可能性減少的預防性治療;及/或受檢者受感染之後,為對抗感染的治療,例如減緩或清除感染或防止其惡化。
微生物抗原可藉由此項技術中熟知的方法製備。舉例而言,該等抗原可藉由製備粗萃取物、藉由部分地純化抗原或藉由重組技術或藉由已知抗原之重新合成,由病毒及細菌細胞直接製備。抗原亦可為編碼抗原肽的核酸形式,該
抗原肽為適於在受檢者中受表現且適於呈現給所免疫受檢者之免疫系統的形式。此外,抗原可為全抗原,或其可為包含至少一抗原決定基之全抗原的片段。
為對抗原併入陽離子性脂質微脂粒內加以改良以及對傳遞至免疫系統之細胞加以改良,可修飾抗原以增強其疏水性或增加抗原上之負電荷。抗原之疏水性可藉由例如與脂質鏈或疏水性胺基酸結合來增強,以便以陽離子性脂質之疏水性醯基鏈使抗原之溶解性改良的同時,維持分子之抗原性質。經修飾之抗原可為胺基酸序列具有經增強之疏水性的脂蛋白、脂肽、蛋白質或肽,及其組合。經修飾之抗原可具有結合於脂質與抗原之間的連接子,例如N-末端α或ε-棕櫚醯基離胺酸可經由二肽絲胺酸-絲胺酸連接子與抗原連接。如下文更詳細所述,與DOTAP/E7調配物相比,DOTAP/E7-脂肽複合物呈現更強的活體內功能性抗原特異性CD8+
T淋巴細胞反應。此外,藉由改變調配緩衝液(其中將抗原封裝在陽離子性脂質複合物內)或藉由使陰離子性部分(諸如陰離子性胺基酸)與抗原共價連接,可操控抗原以增加其負電荷。
如實例1(下文)中所證明,E7抗原之免疫原性藉由共價修飾抗原來增強。可以所得抗原胺基酸序列不存在於抗原所來源之親本蛋白質(parent protein)中的方式將胺基酸序列與抗原共價連接。研究旨在證明,與天然抗原相比,經修飾之抗原可提供更優良的I類MHC結合親和力。正如所證明,此優良的結合親和力轉變為形成針對HPV陽性TC-1
腫瘤的優良活體內抗腫瘤免疫反應。根據以下實例進一步瞭解本發明。
籍由陽離子性脂質之特定劑量組合物有效刺激免疫系統並將抗原傳遞至抗原呈現細胞,從而產生強免疫反應來預防及治療疾病
所有脂質體製備程序中使用細胞培養級水(購自MD Walkersville之Cambrex)或磷酸鹽緩衝鹽水。E7抗原為衍生於HPV 16 E7蛋白(PA Pittsburgh之Molecular Medicine Institute之University of Pittsburgh合成)的H-2Db
限制性CTL抗原決定基(胺基酸49-57,RAHYNIVTF [SEQ. ID. NO. 1])。
該等研究所用的脂質體係使用脂質膜製備。在小玻璃瓶中,藉由(1)將脂質溶於氯仿中;且(2)將氯仿溶液在乾氮氣之穩恆氣流下蒸發來製備脂質膜。藉由將該等膜在真空下保持隔夜來移除痕量之有機溶劑。接著藉由添加水或緩衝液之所要量以使得最終濃度為10 mg/mL來使脂質膜水合12小時。接著將懸浮液於浴槽型超音波儀中超音波處理10分鐘,繼之經由400 nm、200 nm及100 nm過濾膜(購自Hamilton Co., Reno, NV)擠出,且在4℃下儲存。製備
DOTAP/E7時,藉由E7肽之水溶液將脂質膜再水合。亦可使用熟習此項技術者熟知之用於一般脂質體製備的其他方法。
為闡明陽離子性脂質DOTAP之免疫刺激機制及進一步表徵其免疫刺激活性,重要的是評價DOTAP是否可誘導產生適當的Th1細胞因子以進一步增強免疫反應。因此,檢查骨髓源樹突狀細胞("marrow derived dendritic cell,BMDC")在DOTAP刺激之後的細胞因子產生情況。在重組mGM-CSF及mIL-4存在下活體外培養六天之後,在37℃下,將BMDC(每孔2mL中存在106
個細胞)用0.1 μg/mL之對照培養基、DOTAP脂質體、LPD(陽離子性脂質複合之DNA及魚精蛋白(protamine))或脂多醣(LPS)刺激20小時。
藉由BD ELISA組分析上層清液中產生之IL-12及TNF-α。
使用LPD及LPS作為陽性對照。LPD中之細菌DNA含有CpG基元,已知CpG基元可經由鐘樣受體(toll-like-receptor)活化免疫系統並刺激TNF-α。收穫培養液上層清液並藉由ELISA評價IL-12及TNF-α細胞因子量。在圖1中,作為對DOTAP濃度之回應,IL-12產生增加,而非TNF-α產生增加。由此表明,除樹突狀細胞外,其他細胞類型(例如T細胞)亦可能參與所觀測之活體內DOTAP脂質體刺激Th1細胞因子產生。此外,陽離子性脂質不誘導顯著量之促炎性細胞因子TNF-α的事實說明,免疫刺激機制可能不依賴於鐘
樣受體途徑。
在第0天及第7天,用DOTAP/E7調配物將C57BL/6雌性小鼠免疫。使用不同濃度之DOTAP,但以10 μg劑量維持HPV-16 E7抗原之濃度。E7抗原為衍生於HPV 16 E7蛋白的H-2Db限制型CTL抗原決定基(胺基酸49-57,RAHYNIVTF (SEQ. ID. NO. 1))。最後免疫之後第7日,處死小鼠並收穫且解離脾細胞。RBC移除之後,將全部脾細胞群(反應細胞)在完全RPMI-1640培養基中、在40 U/mL重組IL-2(購自MN Minneapolis之R & D systems)存在下、用E7肽(10 μg/mL)刺激5天。活體外擴增CTL之後,反應細胞待用作CTL效應細胞。在該檢定中,TC-1細胞株用作靶細胞。
TC-1細胞為經HPV 16 E6及E7致癌基因及活化H-ras轉型之C57BL/6小鼠肺上皮細胞。為分清效應細胞與靶細胞,將TC-1細胞根據製造商說明書、用PKH-67(購自Sigmaof St. Louis, MO)標記。將效應細胞與標記靶細胞以不同的效應細胞:靶細胞比率(E:T)塗覆在96孔板中,且在37℃下執行溶解反應4小時。接著收穫細胞並用碘化丙啶(propidium iodide,PI)染色以便經由BD FACSCanto數位流式細胞儀(購自CA San Diego之BD Biosciences)分析。E7-特異性溶解百分比係藉由FL 1(PKH-67)陽性區域內PI陽性細胞之百分比來測定。
現轉看圖2,觀測到以100奈莫耳DOTAP接受DOTAP/E7
的小鼠呈現顯著的CTL活性,從而將TC-1細胞特異性殺死,而以600奈莫耳DOTAP接受DOTAP/E7的小鼠展現大幅減小的殺死效應,表明陽離子性脂質之劑量反應效應。
以15奈莫耳DOTAP接受DOTAP/E7的小鼠不產生CTL活性,此與經PBS治療的對照小鼠明顯不同。以HPV E7-陰性BL6細胞作為標靶、藉由培育來源於接受最佳劑量DOTAP之組的效應細胞,可驗證此殺死效應具有E7特異性,且所產生之細胞殺死可忽略不計。除CTL介導之殺死外,亦研究天然殺手("NK")細胞介導之細胞毒性。NK細胞能夠摧毀腫瘤細胞而不經特意免疫或活化且其在先天免疫反應中亦扮演重要角色[Wu, J及Lanier, L L,Natural killer cells and cancer
. Adv Cancer Res 90:127 (2003);及Lodoen, MB,及Lanier, LL,Natural killer cells as an initial defense against pathogens
. Curr Opin Immunol 18:391 (2006)]。以不同劑量之DOTAP接受DOTAP/E7複合物的小鼠呈現針對YAC-1細胞(NK敏感靶細胞)之顯著殺死效應。
為瞭解最佳DOTAP/E7調配物是否誘導T-淋巴細胞(T-細胞)產生且T-淋巴細胞是否有效遷移至表現E7抗原之細胞之位點,對腫瘤浸潤性T淋巴細胞進行免疫組織化學檢查(圖3)。自Charles River Laboratories (Wilmington, MA)購買
6-7週齡C57BL/6雌性小鼠且用於所有動物研究。藉由在第0日將TC-1細胞(105
個細胞)注入小鼠之去毛側腹內來建立皮下HPV-陽性腫瘤。在第6日,將150 μL各含有10 μg之E7肽之DOTAP/E7(15、100、600奈莫耳)之選定調配物皮下注入小鼠(n=6-12)。
在第14日將小鼠之HPV-陽性實體腫瘤切除且解剖,並嵌埋於Tissue-Tek®
OCT化合物(購自CA Torrance之Sakura Finetek)(繼之製備冷凍切片(cryosection))。藉由恆冷切片機(購自H/I Hacker Instruments & Industries Inc.)將樣本切成8 μm厚切片。使用結合FITC之抗-CD8及抗-CD4抗體(購自CA Auburn之Miltenyi Biotec Inc.)測定腫瘤浸潤性T細胞,繼之用DAPI將細胞核對比染色。用Leica SP2共焦顯微鏡獲取切片之影像。
使用TACSTM
TdT套組(購自MN Minneapolis之R & D Systems)執行TUNEL檢定,並根據製造商說明書、用DAB顯像。用Nikon Microphot SA顯微鏡獲取樣本影像。
如圖3中所示,與未經治療之小鼠(圖3A)及接受高劑量DOTAP組合物(600奈莫耳,圖3C)的彼等小鼠相比,以100奈莫耳接受DOTAP/E7的小鼠中存在高比例的CD8+
T淋巴細胞(約5%)(圖3B)。對於CD4+
T細胞得到類似結果(圖3E-3F)。亦對某些腫瘤冷凍切片執行TUNEL檢定以便測定細胞凋亡。在圖3H中,將小鼠用DOTAP/E7複合物中之100nmol劑量脂質治療後,與未經治療之小鼠(圖3G)及高脂質劑量組(圖3I)(呈現正常及致命腫瘤細胞)相比,觀測到彼
等小鼠之腫瘤細胞之濃縮狀及碎片狀細胞核中的TUNEL陽性反應。觀測到陽離子性脂質劑量對T-淋巴細胞產生的劑量反應效應與以上在標題為"陽離子性脂質/抗原複合物以脂質劑量依賴性方式誘導抗原特異性CTL免疫反應"之第3部分中所述的抗原特異性CTL反應之劑量反應效應類似。
圖4展示對小鼠注射含有DOTAP及蛋白質抗原卵白蛋白(ovalbumin)之陽離子性脂質/抗原複合物的結果。對小鼠評價該複合物刺激B-細胞活性及誘導抗體產生之能力,亦名體液免疫反應。體液反應係藉由監測IgG抗體形成來評價。如圖4所說明,DOTAP/卵白蛋白複合物以劑量依賴性方式誘發體液反應。該等資料說明可使用DOTAP調配物刺激細胞免疫反應與體液免疫反應。
據認為調節性T細胞會減弱針對腫瘤相關抗原之T-細胞免疫性且為減緩成功免疫治療及主動接種之主要障礙。因此,研究治療性疫苗調配物中最佳劑量之DOTAP/E7破壞有腫瘤小鼠中由調節性T細胞所維持之免疫耐受性從而增強T-細胞對腫瘤相關抗原之免疫性的能力。用於流式細胞術分析的所有抗小鼠抗體係購自並可購自BD Pharmingen (San Diego, CA)或eBioscience, Inc. (San Diego, CA)。使用1 mg/mL膠原酶(collagenase)及70 μm細胞濾器收穫脾細胞並解離。移除紅血球(RBC)之後,在4℃下,將單細胞懸浮
液用螢光標記之單株抗-CD4(RM4-5)、抗-CD8a(53-6.7)、抗-CD3e(145-2cll)、抗-NK1.1(pk136)及抗-CD25(pc61.5)抗體染色30分鐘。使用Cytofix/CytopermTM
套組(BD Pharmingen)、根據製造商說明書固定並滲透之後,將細胞在4℃下、用50 μl之perm/WashTM
緩衝液中之抗-Foxp3 (FJK-16s)染色30分鐘。將細胞於Perm/WashTM
緩衝液中洗滌兩次以上,且接著最終再懸浮於300 μl染色緩衝液中,且經由BD FACSCanto數位流式細胞儀(購自CA San Diego之BD Biosciences)分析。如圖5中所示,與未經治療之有腫瘤小鼠相比,自接受DOTAP/E7複合物(100奈莫耳DOTAP)之小鼠、而非高劑量(600奈莫耳DOTAP)動物收穫的脾細胞呈現更低量的T-reg細胞亞群(Foxp3+、CD25+及CD25+/Foxp3+)。
在表1中,總結十個用於T-reg分析之代表性實驗。小鼠接受最佳的DOTAP/E7治療之後,脾細胞中CD4+
T細胞群明顯增加,亦發現CD8+
T細胞類似增加(資料未展示)。此外,發現該治療組中T-reg群(CD4+/Foxp3+及CD4+/CD25+/Foxp3+)顯著減少。很顯然,DOTAP/E7在高DOTAP劑量下之抗腫瘤活性降低與T-reg細胞群增加有關。再次觀測陽離子性脂質之劑量反應效應。該結果表明,以最佳脂質劑量用於免疫療法中的陽離子性脂質組合物能夠使藉由破壞有腫瘤小鼠中之耐受性增強所觀測之針對特定癌的免疫反應,並刺激功能性CD4+
及CD8+
抗原特異性T淋巴細胞增殖、同時抑制T-reg細胞群成為可能。
在圖6中,在第0日將TC-1細胞皮下注入小鼠中,以便誘導HPV-陽性腫瘤生長。在第6日,小鼠在腹部之對側皮下接受含有10 μg E7肽之DOTAP/E7組合物。複合物中DOTAP脂質濃度係自3奈莫耳至600奈莫耳不等(3、15、30、75、150、300及600奈莫耳)。第23日,與未經治療對照組相比,低劑量之DOTAP(15奈莫耳)展現部分腫瘤抑制效應(P<0.05),而30、150或300 nmol之DOTAP呈現經增強之功效(P<0.01)。75 nmol之DOTAP展現最顯著之腫瘤消退效應(P<0.001)。此外,給與高劑量DOTAP(600 nmol)之小鼠未展現抗腫瘤活性,證明高劑量之DOTAP脂質體可能對免疫反應誘導負調節。此外,100奈莫耳劑量、但無E7肽的DOTAP脂質體,對腫瘤生長未展現明顯抑制,說明抗腫瘤效應具有抗原特異性。
對未處理(naive)小鼠(n=4-6)注射PBS對照物(圖7A、7C及7E)或含有100 nmol總脂質、具有0.5% 1-油醯基-2-[6-[(7-硝基-2-1,3-苯并噁二唑-4-基)胺基]己醯基]-3-三甲基銨丙烷]("NBD-DOTAP")的DOTAP/E7(圖7B、7D及7F)。在注射後第4小時,製備引流淋巴結細胞並用針對表面標記之適當抗體染色。對全部淋巴結細胞內NBD與CD11c(圖7A及7B)或CD86(圖7C及7D)之共表現進行分析,而對CD3+
群內NBD與CD8(圖7E及7F)之共表現進行選通(gate)並分析。
數字表示象限內細胞之百分比。在注射後之不同時點收穫並藉由流式細胞術分析引流淋巴結細胞。在NBD-DOTAP/E7注射之後第4小時,與未經治療小鼠相比,全部淋巴結細胞中CD11c+
細胞之數目增加2.5倍以上(圖7A及7B)。研究NBD+
細胞上共刺激分子CD86之表現,且證明NBD+
細胞具有高含量之CD86(圖7C及7D),說明皮下注射DOTAP可誘導樹突狀細胞活化。亦藉由用抗-CD3、抗-CD4及抗-CD8抗體共染色來研究諸如T淋巴細胞之其他細胞類型對NBD的吸收。選通並分析CD3+
細胞(圖7E及7F),且表明注射NBD-DOTAP之後,無NBD被吸收。結果清楚地證明,免疫不久之後,NBD-DOTAP主要被樹突狀細胞吸收(約80%),且DOTAP誘導活化樹突狀細胞遷移至引流淋巴結,引起樹突狀細胞-T細胞相互作用並誘發強T-細胞反應。
已知活化T細胞或NK細胞所分泌的IFN-γ在Th1型免疫反應以及誘導CTL反應中扮演重要角色。為評價DOTAP/E7接種所誘導的功能性CD8+
T淋巴細胞是否能夠產生基本細胞因子,在最後免疫之後一週時分離對照小鼠或免疫小鼠之脾細胞並用5 μg/ml E7肽或不用肽培育6小時,繼之將IFN-γ進行細胞內染色。如圖8中所示,與陰性對照組相比,在接受以最佳脂質劑量之DOTAP調配之E7及LPD陽性對照物的小鼠中,產生IFN-γ之CD8+
細胞數目明顯更高。
CD8+
細胞產生IFN-γ係以E7特異性方式發生。該等結果表明,最佳劑量之DOTAP對於誘導CTL以及在全身淋巴器官中形成產生IFN-γ之CD8+
T淋巴細胞而言為強疫苗佐劑。
為比較陽離子性脂質/E7調配物與其他佐劑誘導針對腫瘤之免疫反應的功效,在建立腫瘤之後第六天,每種調配物治療6-12個有腫瘤小鼠,經E7肽調配之脂質體包含:(1)陽離子性脂質為100奈莫耳劑量組成的陽離子性脂質(DOTAP、DOEPC及DOTMA);(2)陰離子性脂質(DOPG);或(3)佐劑(完全弗氏佐劑(complete Freund adjuvant;"CFA")或CpG ODN1826)。在圖9中,與對照組相比,在第26日,接受佐劑與陽離子性脂質調配物的小鼠對腫瘤生長
展現有效的抑制。接受陰離子性脂質DOPG/E7的小鼠不展現腫瘤消退。與接受CpG/E7或CFA/E7調配物的彼等小鼠相比,接受最佳DOTAP/E7及DOEPC調配物之小鼠呈現更優的抗癌活性(p<0.01)。與接受CpG/E7或CFA/E7調配物的彼等小鼠相比,接受最佳DOTMA/E7之小鼠亦呈現更優的抗癌活性。
在注射之後第2小時,將接受含有0、15、100或600 nmol脂質之DOTAP/E7之皮下注射之小鼠(每種條件下n=4)的引流淋巴結分離。在流式細胞術分析之前,將全部淋巴結細胞在37℃下、用DCFH-DA化合物培育30分鐘。選通大顆粒細胞並分析ROS表現,其中螢光產物DCF在ROS存在下產生。具有陽性ROS信號之細胞的相對百分比列於圖10A中。引流淋巴結中之細胞毒性係藉由在注射DOTAP/E7之後第10小時收集細胞並用適當抗體染色來執行。在圖10B中,樹突狀細胞(DC)中死細胞之相對百分比係以線圖()展示且柱狀圖表示每105
個LN細胞中活DC之數目。將數字依照未經治療之對照組歸一化且進行統計分析(**p<0.01)。接受15 nmol DOTAP之小鼠的細胞呈現ROS產生之基底量(<5%),而注射100奈莫耳劑量DOTAP之小鼠的細胞產生相對較高量之ROS(約20%)。驚人地,來自DOTAP 600 nmol組的大部分大顆粒細胞(約80%)展現陽性ROS信號。在皮下注射後第10小時,收穫全部淋巴結細
胞並藉由流式細胞術分析細胞死亡(圖10B)。CD11c+
(樹突狀細胞)群內細胞死亡之百分比(碘化丙啶陽性)隨脂質劑量而增加,並與圖10A中所示之ROS產生呈正相關關係。對於DOTAP高劑量注射組,與未經治療之對照組相比,DC群中細胞死亡之百分比高達約兩倍。圖10B中亦展示,每105
個淋巴結細胞中活CD11c+
細胞之數目隨DOTAP脂質劑量而變化。在其他治療組中,接受具有100 nmol脂質之DOTAP/E7的小鼠呈現最高量(**p<0.01)之活樹突狀細胞。
小鼠接受最佳調配物之後第2天,將引流淋巴結稱重。總之,結果表明高劑量DOTAP誘導之ROS產生可能會促使樹突狀細胞死亡。
此外,如圖10C中所說明,陽離子電荷密度對於免疫刺激效應及所產生之抗癌活性很重要。藉由將惰性、中性脂質DOPC與陽離子性脂質/E7複合物高比率共調配從而降低陽離子電荷密度來減少ROS形成與抗癌活性。
如上所述製備陽離子性脂質/抗原複合物。使用LavaPepTM
肽量化套組(購自Australia Sydney之Fluorotechnics)、以脂質體結合肽之百分比測定肽封裝效率。由於未合併之脂肽聚集並無法通過排除過濾器,因此所合併之脂肽係在1%SDS存在下、以與所擠出之脂質體相關之量量測且報導為平均值±SD(n=3)。對於水溶性肽,諸如天然E7及KSS-E7,藉由Microcon®
離心過濾裝置(購自MA Bedford之
Millipore)將未結合肽與複合物分離。未結合肽之濃度係使用LavaPepTM
、根據製造商說明書量測。封裝效率係以(1-未結合肽%)測定且作為平均值±SD(n=3)報導於表2中。
在(腫瘤建立之後)第6日,用DOTAP/E7(含有10 nmol或5 nmol肽)或DOTAP/E7-脂肽調配物(含有5 nmol肽抗原)對具有TC-1腫瘤之小鼠(每組8-12個)給與單劑治療。在每種情況下,使用100奈莫耳陽離子性脂質之組合物。具有5 nmol肽的DOTAP/E7不展現明顯的抗腫瘤活性。相反,具有5 nmol抗原之E7-脂肽(α或ε位置)當調配於DOTAP脂質體中時,與具有5 nmol天然E7抗原之DOTAP相比,均展現明顯增強的治療效應(**p<0.01)(圖11)。脂肽誘發之抗腫瘤活性與具有10 nmol肽之DOTAP/E7所誘發之抗腫瘤活性類似。為棕櫚醯化E7肽(無KSS間隔子)的PA-E7當調配於DOTAP脂質體中時,未展現如其他脂肽調配物所顯現之強
抗腫瘤活性,可能是因為抗原決定基因脂肪酸與肽直接連接而被隱藏。DOTAP/E7-脂肽之抗腫瘤活性至少部分地因抗原之封裝效率提高而增強。此證明,藉由增強抗原之疏水性,可增強在複合物內之合併,從而增強抗原特異性免疫反應。經右旋糖(5%)治療之小鼠用作陰性對照。
用DOTAP/E7-脂肽免疫,增加分泌IFN-γ之CD8+
T細胞之產生。將不同調配物注入小鼠,且在最後免疫之後第7日,將免疫小鼠的脾細胞分離。將細胞用或不用E7肽(5 μg/ml)活體外刺激6小時,且在FACS分析之前,用表面CD8標記及細胞內IFN-γ細胞因子染色。各治療組每104
個全CO8+
中CD8+
IFN-γ+
雙陽性細胞之百分比係以平均值±SD展示於圖12中且藉由配對t-檢驗進行統計分析(**p<0.01,n=4)。
為評價DOTAP/E7-脂肽接種所誘導之抗原決定基特異性免疫反應,分析產生IFN-γ之CD8+
T細胞。在最後免疫之後一週時,將對照小鼠或免疫小鼠之脾細胞分離,且用5 μg/ml E7肽或不用肽刺激,繼之對IFN-γ進行細胞內染色。
如表2(上文)中所述及圖12中所示,在脂肽抗原之情況下,抗原在複合物內之合併明顯改良,且與接受10 nmol與5 nmol之天然E7之彼等小鼠相比,在接受調配於DOTAP脂質體中之5 nmol之ε-PA-KSS-E7的小鼠中,產生IFN-γ之CD8+
細胞之數目因而明顯更高(**p<0.01)。此外,在相等
抗原量下,KSS-E7展現比天然E7更優良的結果。CD8+
細胞產生IFN-γ具有E7特異性,因為未衝擊細胞僅展現背景量之細胞因子。該等結果表明,將具有經增強之疏水性及複合效率之E7-脂肽併入DOTAP脂質體,可明顯增強陽離子性脂質/抗原複合物之功效並使淋巴器官中產生IFN-γ之CD8+
T淋巴細胞的量提高。
不連接脂質鏈的KSS(離胺酸-絲胺酸-絲胺酸)-E7肽當調配於DOTAP陽離子性脂質中時,與調配於DOTAP複合物中之天然E7肽相比,儘管其封裝效率可與天然E7之封裝效率相比擬(表2),但得到強得多的免疫反應(圖11及12)。已知肽結合且穩定I類MHC分子的能力可與其誘導特異性CTL反應的能力直接相關[Feltkamp, M. C.等人,Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells
. Eur J Immunol, 23, (9), 2242-2249 (1993)]。評價天然E7肽及KSS-延長E7肽之I類MHC結合親和力。在27℃下,將濃度為5×105
個細胞/毫升的RMA-S細胞(小鼠淋巴瘤細胞株)用天然E7或KSS-E7肽(10 μM)培育隔夜。用培養基培育之細胞用作對照。接著將細胞在37℃下培育2小時。洗滌後,將細胞用抗細胞表面上之H-2Db
或H-2Kb
分子的螢光結合單株抗體染
色,再進行流式細胞術分析。E7肽(胺基酸49-57)為限於H-2Db
的已知抗原決定基,且與對照組相比,其引起RMA-S細胞上H-2Db
分子之四倍上調(圖13)。關於KSS-E7肽觀測到平均螢光值提高8倍。用E7或KSS-E7肽培育之後,經偵測,RMA-S細胞上之H-2Kb
分子未上調。結果證明,與天然E7肽相比,KSS-E7對H-2Db
分子具有改良之結合親和力,其當調配於DOTAP/抗原複合物中時產生優良的抗腫瘤活性。
研究證明,免疫原性肽之免疫原性可藉由連接與免疫原性肽所來源之天然親本蛋白質序列無關的胺基酸短序列來改良或改變。
對於本研究,將DOTAP溶於氯仿中,且在16×100 mm玻璃管中將7 mg DOTAP製成乾膜。在真空下乾燥之後,在(1)高離子濃度環境(15 mM磷酸鈉、150 mM NaCl,pH 7.0)下,或(2)低離子濃度環境(0.5 ml抗原,於15 mM磷酸鈉、50 mM NaCl中,pH 7.0)下,將脂質體用0.5 ml抗原(HPV-16 E7,胺基酸11-20,YMLDLQPETT(SEQ. ID. NO. 2))水合。粒徑為100 nm。
藉由將顆粒經100K Nanosep微過濾器(購自MI Ann Arbor之Pall Corp.)、在微離心管中以5000 rpm過濾20分鐘來分析抗原封裝效率。藉由逆相層析法分析起始緩衝液中及Nanosep過濾器之濾液(flow-through)中之抗原濃度,以測
定封裝百分比。如表3所示,封裝效率係以(1-未合併肽%)計算。
在高離子濃度環境下,離子相互作用降至最小。更高離子濃度環境有效減少負電荷。藉由增加調配緩衝液(150 mM NaCl)之離子濃度,帶負電荷之抗原與帶正電荷之脂質之間離子相互作用減至最小,使封裝減少。藉由降低離子濃度,脂質與抗原之間的離子相互作用增強,使抗原在複合物內之合併增加。併入陽離子性脂質/抗原複合物內之效率因而可如下增強:藉由改變調配緩衝液或藉由將陰離子性或聚陰離子性化合物與抗原連接來操控抗原增加負電荷。
如美國專利第7,303,881號中所述,廣泛類別之陽離子性脂質可連同抗原一起起強免疫刺激劑之作用,以在疾病治療中產生抗原特異性免疫反應。舉例而言,美國專利7,303,881揭示,如依據陽離子性脂質刺激DC2.4樹突狀細
胞上共刺激分子CD80/CD86之表現(圖14A及14B)所證明,包含陽離子性脂質的脂質體將樹突狀細胞活化。如圖14A中所示,不同陽離子性脂質體刺激DC2.4細胞上CD80/CD86之表現的能力大不相同。脂質體轉染劑RTM (Lipofectamine. RTM)(聚陽離子性脂質2,3-二油醯氧基-N-[2(精胺甲醯胺基)乙基]-N,N-二甲基-1-丙銨三氟乙酸鹽(DOSPA)與中性脂質二油醯基磷脂醯乙醇胺(DOPE)之3:1(w/w)脂質體調配物)及由O,O'-二肉豆蔻基-N-離胺醯基天冬胺酸酯(DMKE)及O,O'-二肉豆蔻基-N-離胺醯基-麩胺酸酯(DMKD)製成之脂質體可強烈刺激CD2.4細胞表現CD80/CD86。
不同陽離子性脂質刺激DC2.4細胞上CD 80之表現的能力不同。脂質之親水性頭與親脂性尾皆對此能力有明顯的影響。舉例而言,具有乙基磷酸膽鹼(EPC)頭基團之DXEPC脂質似乎通常比具有三甲基銨丙烷(TAP)頭基團之DXTAP脂質更強。在具有一特定頭基團結構的脂質內,具有更短醯基鏈之脂質(1,2-二月桂醯基-sn-甘油基-3-乙基磷酸膽鹼(DLEPC-12:0)、1,2-二肉豆蔻醯基-sn-甘油基-3-乙基磷酸膽鹼(DMEPC-14:0))或具有不飽合醯基鏈之脂質(1,2-二油醯基-sn-甘油基-3-乙基磷酸膽鹼(DOEPC-18:1))似乎比具有更長醯基鏈之彼等脂質(1,2-二棕櫚醯基-sn-甘油基-3-乙基磷酸膽鹼(DPEPC-16:0))或具有飽和醯基鏈之脂質(1,2-二硬脂醯基-sn-甘油基-3-乙基磷酸膽鹼(DSEPC-16:0))更強(圖14B)。該等資料證明,多種陽離子性脂質能
夠刺激樹突狀細胞之活化。因此其他研究選擇三種代表性的陽離子性脂質DOTAP、DOTMA及DOEPC測定陽離子性脂質起免疫刺激劑作用之機制。本文中就代表性陽離子性脂質所提供之資料可外推至刺激免疫反應的其他陽離子性脂質。
本發明研究資料已形成以下觀測結果:陽離子性脂質不僅為抗原有效靶向及傳遞至免疫系統之APC的媒劑,而且在低劑量組成範圍下起強佐劑之作用,以經由MAP激酶依賴性信號傳導途徑之活化、促使包括細胞因子及趨化因子在內的免疫系統調節性分子產生來直接影響免疫系統功能。陽離子性脂質對調配物之免疫刺激能力之明顯的劑量-反應效應業已證明。為此,已證明,當將抗原(諸如16型HPV之E7蛋白之肽抗原決定基)封裝於DOTAP、DOTMA或DOEPC之陽離子性脂質體內(DOTAP/E7、DOTMA/E7或DOEPC/E7)且藉由單次皮下注射將抗原/脂質複合物以約100奈莫耳劑量組成投藥時,其誘導小鼠中HPV 16 E7-陽性腫瘤TC-1之消退。接受脂質/抗原複合物後,顆粒主要被樹突狀細胞(主要的專門抗原呈現細胞)吸收。正如所證明,樹突狀細胞開始活化並遷移至引流淋巴結,促成針對抗原特異性TC-1腫瘤之免疫反應。小鼠在接受DOTAP/E7注射後產生功能性CD8+
T淋巴細胞,且由於腫瘤微環境中浸潤性T細胞之數目增加而腫瘤尺寸減小並呈現增強之細胞凋亡。所得鐘形陽離子性脂質劑量反應曲線證明極低劑量下之活性(高於及低於最佳劑量時,活性減小),說明作
為佐劑或免疫刺激劑之陽離子性脂質之活性係如此之強以致每次注射之EC50
低至約15 nmol。高劑量之陽離子性脂質使免疫刺激活性消失。亦已證明,當將抗原(諸如卵白蛋白)併入陽離子性脂質體內且以單次皮下注射投藥時,產生針對抗原之有效抗體。顯然,在最佳劑量組成下,陽離子性脂質及陽離子性脂質/抗原複合物提供適用於預防及治療疾病之簡單、安全且極其有效的免疫療法。
調節性T細胞最初由Gershon等人[Eardley, D D等人,Immunoregulatory circuits among T-cell sets. I. T-helper cells induce other T-cell sets to exert feedback inhibition
. J Exp Med 147:1106;及Cantor, H等人,Immunoregulatory circuits among T-cell sets. II. Physiologic role of feedback inhibition in vivo: absence in NZB mice
. J Exp Med 147:1116 (1978)]描述於十九世紀七十年代且稱之為抑制性T細胞。最新研究已於若干鼠模型以及癌症患者中探究出CD4+CD25+調節性T細胞(T-reg)在抑制腫瘤免疫性中的角色[Comes, A等人,CD25
+regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine
. J Immunol 176:1750 (2006)]。
在癌症患者之周圍血液中,T-reg細胞群之出現率增加[Sasada, T等人,CD4
+CD25
+regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression
. Cancer 98:1089 (2003)]。其亦富集於腫瘤浸潤性淋巴細胞
中及引流淋巴結中[Baecher-Allan, C及Hafler, DA,Suppressor T cells in human diseases
. J Exp Med 200:273 (2004)]。此外,T-reg在腫瘤相關組織中之積累預示不良的預後或存活率[Baecher-Allan, C及Anderson, DEImmune regulation in tumor-bearing hosts
. Curr Opin Immunol 18:214 (2006)]。儘管T-reg係如何減弱正常T-細胞免疫性之詳細機制尚不十分瞭解,但已報導藉由使用抗-CD25抗體阻斷T-reg細胞來增強抗腫瘤活性[Attia, P,等人,Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with Melanoma
. J Immunother 28:582 (2005)]。實際上,顯然需要對接受候選人類腫瘤疫苗之患者監測並表徵效應細胞與T-reg反應[Baecher-Allan, C及Anderson, DEImmune regulation in tumor-bearing hosts
. Curr Opin Immunol 18:214 (2006)]。
在本發明研究中,發現DOTAP/E7之抗腫瘤活性與T-reg細胞之減少有明顯關聯。
陽離子性脂質因此構成新穎類別之免疫刺激劑,其在特定劑量下可有效應用於調節身體免疫反應及開發供預防性使用與治療性使用的治療劑及疫苗。
數年來已知蛋白質及肽就用於開發免疫療法及疫苗而言具有顯著的潛力。成功開發該等療法之主要障礙為抗原至免疫系統的無效傳遞。此外,已加強研究開發用於傳染病及用作癌症療法之安全的強免疫刺激劑。涉及佐劑(諸如
獲自SEPPIC, Inc.的Montanide ISATM
51及720,其為來自二縮甘露醇單油酸酯(mannide monooleate)家族之界面活性劑分別與礦物油或非礦物油之混合物)的現行技術已以針對人類之傳染病及癌症的疫苗得到測試[Aucouturier, J等人,Montanide ISA 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines
. Expert Rev Vaccines 1:111 (2002)]。各種研究證明了Montanide ISATM
51及720增強抗體效價以及特異性CTL反應的能力[Yamshchikov, GV等人,Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients
. Int J Cancer 92:703 (2001)]。
然而,此類型油包水佐劑需要在製備過程中進行乳化之關鍵步驟,此步驟並不總是易於控制。更重要地是,與Montanide ISA 720組合之多抗原決定基多肽TAB9的I期臨床試驗表明,低劑量組之八名自願者中有七名展現中度或嚴重的局部炎症,而高劑量組之八名受檢者中有四名顯現肉芽腫及無菌性膿腫[Toledo, H等人,A phase I clinical trial of a multi-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers
. Vaccine 19:4328 (2001)]。ISA 720係由代謝性油狀物組成且據推測對人類具有更小的反應原性。然而,如同其他大部分佐劑,乳液中之界面活性劑可能會激發樹突狀細胞及巨噬細胞上之鐘樣受體(TLR),從而誘導產生NF-kB及炎性反應[Aucouturier, J等人,Montanide ISA 720 and 51 : a new generation of water in oil emulsions as adjuvants for human vaccines
. Expert Rev Vaccines 1:111 (2002)]。
已證明陽離子性脂質不增強NF-κB之表現,說明陽離子性脂質刺激樹突狀細胞係經由NF-κB獨立機制進行信號傳導[Cui, Z.等人(2005).Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier. Mol Pharm 2: 22-28)
]。因此,陽離子性脂質體可能屬於具有改良之安全分布的獨特類佐劑。
亦已證明,藉由將抗原脂質化,諸如將單棕櫚酸與KSS-延長E7肽(在α或ε位置)連接,可改良肽封裝於陽離子性脂質體/抗原複合物內之效率(與水溶性抗原之封裝效率相比)。由僅兩個分子組成的DOTAP/E7-脂肽複合物誘導抗原特異性CTL之產生總體增強,以便消除HPV陽性TC-1細胞。將陽離子性脂質組成維持在100 nm。當將抗原以低劑量(5 nmol或小於5 nmol)投與有腫瘤小鼠時,與全劑量(10 nmol)之初始DOTAP/E7調配物相比,DOTAP/E7-脂肽複合物呈現優良的抗腫瘤活性。脂肽之增強之抗原性及抗腫瘤活性係與脂肽於脂質體中之增強之封裝有關。如表2中所示,在2.5 mol%總脂質之肽負荷下,脂肽之俘獲效率為90%,而天然水溶性E7肽中僅約25%併入複合物內。類似經增強之脂肽脂質體封裝亦已報導[Yagi, N.等人,Preparation of functinal liposomes with peptide ligands and their binding to cell membranes
. Lipids, 35, 673-680
(2000);及Liang, M. T.等人,Encapsulation of lipopeptides within liposomes: effect of number of lipid chains, chain length and method of liposome preparation
. Int J Pharm, 301, 247-254 (2005)]。為藉由I類MHC途徑呈現肽,肽須進入APC之細胞質內。陽離子性脂質體將經封裝之肽、而非游離肽傳遞至細胞內。因此,肽封裝愈高,則可預期傳遞及抗原性愈佳。此外,在所證明的給藥條件下,陽離子性脂質亦作為強免疫刺激劑起作用,產生引起所觀測到之腫瘤細胞死亡的強抗原特異性免疫反應。
ROS之重要性涉及先天免疫反應及T細胞活化[Kantengwa等人,Superoxide anions induce the maturation of human dendritic cells
. Am J Respir Crit Care Med 167:431-437 (2003)],且高ROS產生引起細胞死亡[Tobiume K等人,ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis
. EMBO Rep 2:222-228 (2001)],[Aramaki Y等人,Induction of apoptosis in WEHI 231 cells by cationic liposomes
. Pharm Res 17:515-520 (2000)]。已證明陽離子性脂質在引流淋巴結之細胞中誘導ROS且高劑量之DOTAP脂質引起樹突狀細胞被殺死。實際上,Iwaoka等人已證明陽離子性脂質體可在活體外巨噬細胞中誘導ROS[Iwaoka S等人,Cationic liposomes induce apoptosis through p38 MAP kinasecaspase-8-Bid pathway in macrophage-like RAW264.7 cells
. J Leukoc Biol 79:184-191 (2006)]。圖10A中所示之資料明顯
證明,在皮下注射DOTAP/E7複合物之後,陽離子性脂質能夠在引流淋巴結中活體內產生ROS。上述資料亦說明,高劑量之陽離子性脂質所產生的過量ROS致使樹突狀細胞死亡增加。該等資料支持將陽離子性脂質以足以誘導受檢者免疫系統之細胞中產生ROS的劑量投與受檢者,其中所誘導之ROS量係足以使免疫反應強於在陽離子性脂質不存在時所呈現之免疫反應。由於DOTAP/E7複合物中之高DOTAP劑量,因此可能存在與免疫刺激作用下降及所得抗癌活性下降相關的其他可能原因。然而,DLN中減少量之活化APC在高疫苗劑量下所觀測到之淋巴細胞浸潤(圖3)、抗原特異性CTL活性及IFN-γ產生(圖2及圖8)以及(最重要地)抗腫瘤活性(圖6)之下降中無疑扮演著重要角色。另一方面,如下文在實例2中所述,由於DOTAP誘導之ROS可能係介導為疫苗活性所必需之隨後信號轉導的起始信號,諸如ERK及p38 MAP激酶,因此需要產生所要量之ROS。
此外,在圖10C中已證明陽離子電荷密度對免疫刺激效應及所得抗癌活性的重要性。本文中已證明,藉由將惰性、中性脂質DOPC與陽離子性脂質/E7複合物高比率共調配從而降低陽離子電荷密度來減少ROS形成與抗癌活性。
實例1證明陽離子性脂質劑量對免疫刺激效應的重要性且亦強調陽離子性脂質/抗原複合物在開發產生強抗原特異性免疫反應以便治療癌症及多種疾病(諸如細菌感染及病毒感染)之簡單且安全免疫療法中的作用。
陽離子性脂質之免疫刺激活性之機制:MAP激酶ERK之磷酸化及趨化因子之誘導
TC-1細胞係由TC Wu(Johns Hopkins University, Baltimore, MD)提供。該等細胞為已經HPV 16 E6及E7致癌基因及活化H-ras轉型之C57BL/6小鼠肺內皮細胞。將細胞生長於補充有10%胎牛血清及100 U/ml青黴素及100 mg/mL鏈黴素之RPMI培養基(購自CA Carlsbad之Invitrogen)中。源於HPV 16 E7蛋白的I類MHC限制肽(胺基酸49至57,RAHYNIVTF [SEQ. ID. NO. 1])係由University of Pittsburgh Peptide Synthesis Facility使用Advanced ChemTech 200型肽合成器、藉由固態合成法合成並藉由HPLC純化[如Feltkamp等人,Eur J Immunol
23, 2242-2249 (1993)中所述]。特異於pERK及ERK2及siRNA的小鼠單株抗體係購自CA Santa Cruz之Santa Cruz Biotechnology, Inc.。特異於磷酸化p38(p-p38)的兔多株抗體係獲自MA Danvers之Cell Signaling Technology Inc.。GeneChip小鼠基因組430 2.0陣列獲自Affymetrix, Inc.。PD-98059、U-0126、PP2、渥曼青黴素(wortmannin)及GF109203X獲自Calbiochem。百日咳毒素(Pertussis Toxin)及其他試劑獲自Sigma。
所有脂質購自Avanti Polar Lipids (Alabaster AL)。小的單層DOTAP、DOEPC及DOTMA脂質體係藉由薄膜水合、
繼之擠出來製備。將氯仿中具有或不具有藥物之脂質在玻璃管中、在氮氣流下乾燥為薄層。將薄膜真空乾燥2-3小時且接著於含有E7肽的細胞培養級水(購自MD Walkersville之Cambrex)或緩衝液中再水合至最終濃度為每毫升0.7毫克脂質及0.1毫克E7(莫耳比=11:1)。將脂質分散液依序經由具有0.4、0.2及0.1 μm之孔徑之聚碳酸酯膜擠出。未俘獲肽不移除。將脂質體在4℃下儲存直至使用。E7肽與脂質體之結合係藉由量測脂質體結合肽之百分比測定。簡而言之,藉由Microcon®
離心過濾裝置(Millipore, Bedford, MA)將未結合E7肽與DOTAP/E7或DOTAP/E7/藥物(MAP激酶抑制劑)複合物分離,且藉由Micro BCATM
蛋白質檢定套組(Pierce, Rockford, IL)量測未結合肽之濃度。肽結合效率係以未結合肽百分比測定。約30%之E7肽與脂質體結合。
數據係以至少3個獨立實驗之平均值±SD提供。使用雙尾學生t檢驗評價均值差之統計顯著性。顯著性設定在p<0.05。
藉由獲自MD Germantown之Qiagen之RNeasy Mini套組、根據製造商說明書萃取RNA。使用7 μg之總RNA合成cDNA。該反應結合T7-(dT)24
引子使用獲自MD Gaithersburg 之Life Technologies的定製cDNA套組。接著,使用BioArray高產率RNA轉錄套組、經由cDNA反應產生經結
合生物素之cRNA。接著,將cRNA於破碎緩衝液(5X破碎緩衝液:200 mM Tris-乙酸鹽(pH 8.1)、500 mM KOAc、150 mM MgOAc)中、在94℃下破碎35分鐘後再進行晶片雜交。接著將15 μg碎裂cRNA添加至雜交混合物(0.05 μg/μl碎裂cRNA;50 pM對照寡核苷酸B2、BioB、BioC、BioD
及cre
雜交對照物;0.1 mg/mL鯡魚精DNA;0.5 mg/mL乙醯化BSA;100 mM MES;1 M NaCl;20 mM EDTA;0.01% Tween 20)中。使用10 μg之cRNA進行雜交。使陣列在GeneChip 640雜交反應培養箱中、在45℃下雜交16小時。洗滌陣列並於GeneChip Fluidics Station 400中、用R-藻紅素抗生蛋白鏈菌素(R-phycoerythrin streptavidin)染色。之後,用Hewlett Packard基因陣列掃描儀掃描陣列。
洗滌、掃描及基本分析均使用Affymetrix GeneChip微陣列程式組5.0軟體。樣本品質係藉由檢查某些基因之3'與5'端強度比來評價。
使用改自以下文獻中所述的程序產生BMDC:Inaba K.等人,Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor
. J Exp Med 176: 1693-702, (1992)。簡而言之,自C57BL/6雌性小鼠之股骨及脛骨收穫骨髓。在紅血球溶解之後,塑性黏附(plastic adherence)容許樹突狀細胞前驅細胞與淋巴細胞分離。將剩餘骨髓細胞於補充有10% FBS、非必需胺基酸、抗生素
化合物(antibiotic/antimycotic)及GM-CSF及1L-4(R & D systems)(各自為1,000 U/ml)的RPMI 1640培養基中培養6天。隔日更換培養基。在第6日,將細胞用於檢定。如對CD11c表現使用流式細胞術所證實,該等細胞90%以上為樹突狀細胞。
藉由獲自Qiagen之Rneasy Mini套組、根據製造商說明書分離BMDC中的總RNA。在37℃下用去氧核糖核酸酶(Dnase)消化30分鐘之後,藉由分光光度計測定總RNA濃度。小鼠CCL2、CCL3、CCL4及β-肌動蛋白之引子對(對於CCL2,前置引子5'-AAGCCAGCTCTCTCTTCCTC-3'[SEQ. ID. NO. 3]及反置引子5'-CCTCTCTCTTTGAGCTTGGTG-3'[SEQ. ID. NO. 4];對於CCL3,前置引子5'-ATCATGAAGGTCTCCACCAC-3'[SEQ. ID. NO. 5]及反置引子5'-TCTCAGGCATTCAGTTCCAG-3'[SEQ. ID. NO. 6];對於β-肌動蛋白,前置引子5'-GCTCTGTGCAAACCTAACCC-3'[SEQ. ID. NO. 7]及反置引子5'-TGTGATGGTGGGAATGGGTCAG-3'[SEQ. ID. NO. 8]及反置引子5'-TTTGATGTCACGCACGATTTCC-3'[SEQ. ID. NO. 9])[Wang H等人,Potential involvement
of monocyte chemoattractant protein (MGP)-l/CCL2
in IL-4-mediated tumor immunity through inducing dendritic cellmigration into the draining lymph nodes
, Int Immunopharmacol 3:627-42, (2003)]係藉由IDT技術合成。
使用一步法RT-PCR套組(購自CA Carlsbad之Invitrogen)執
行RT-PCR,其中逆轉錄及DNA擴增在同一反應中發生。
簡而言之,使用1 μg之總RNA作為反應模板,其包括在逆轉錄酶與Taq聚合酶存在下的適當引子。將混合物在45℃下培育30分鐘,且將在94℃下30秒、在55℃下30秒及在72℃下2分鐘循環25次。擴增適當的陰性對照包括逆轉錄酶不存在下的反應。PCR產物係藉由紫外光透照(UV transillumination)經溴化乙錠染色之1.5%瓊脂糖凝膠來目測。
在C57BL/6雌性小鼠(n=5)接受PBS、DOTAP/E7或DOTAP/E7/MAP激酶抑制劑之皮下注射之後第2天,藉由CCL2 ELISA套組(購自BD Biosciences)分析引流淋巴結內的CCL2產生量。將C57BL/6小鼠之BMDC如所述、在重組mGM-CSF及mIL-4存在下培養六天。在第6日,將BMDC(1毫升/孔中存在106
個細胞)在37℃下、用對照培養基或脂質體刺激1小時、24小時或48小時。對於抑制劑處理,將抑制劑與BMDC一起預培育20分鐘後再進行脂質體刺激。藉由CCL2 ELISA套組分析上層清液中的CCL2產生。
在第2日移除小鼠之引流淋巴結,嵌埋於OCT化合物中,快速冷凍於液氮中,且在-80℃下儲存直至供免疫組織化學分析使用。山羊ABC染色系統(購自CA Santa Cruz之Santa Cruz Biotechnology Inc.)係根據製造商方案使用。
簡而言之,剪切引流淋巴結之8 μm厚冷凍切片並在冷丙酮
中固定10分鐘且於PBS中洗滌3次,每次洗滌5分鐘。接著將切片依序在1%過氧化氫(於H2
O中)培育10分鐘以阻斷內源過氧化物酶、在PBS中之1.5%阻斷血清(驢血清)中培育60分鐘、接著在4℃下與一次抗體(1:100稀釋;購自CA Santa Cruz之Santa Cruz Biotechnology Inc.)一起培育隔夜。接著用AB酶試劑將載片與經結合生物素之二次抗體(1:150稀釋;購自CA Santa Cruz之Santa Cruz Biotechnology Inc.)一起培育30分鐘,且用3滴過氧化物酶底物培育10分鐘或更久。
在不同處理之後收集BMDC細胞溶胞液,且將總細胞蛋白質於聚丙烯醯胺/SDS凝膠上解析且接著轉移至聚偏二氟乙烯膜上。將膜用Tris-緩衝鹽水中之5%脫脂乳阻斷1小時且接著與一次抗體一起培育1小時至隔夜。用Tris-緩衝鹽水將膜洗滌三次後,將膜用結合辣根過氧化物酶之二次抗體培育1小時。使用ECL Plus (Amershan International)、繼之使用自動射線照相術、藉由強化學螢光偵測與蛋白質條帶相關的過氧化物酶活性。
使用脂質體轉染劑2000(Lipofectamine 2000)試劑(購自CA Carlsbad之Invitrogen)、根據製造商說明書、用對於ERK1之siRNA及對照siRNA(購自CA Santa Cruz之Santa Cruz Biotechnology)執行RNA干擾實驗。簡而言之,在第5日,將BMDC以每孔5×105
個接種於12孔板中且立即使用
4 μl轉染試劑、用80 nM siRNA轉染。將細胞用DOTAP或LPS另培育16小時。藉由ELISA分析上層清液之CCL2。
藉由使用Affymetrix微陣列分析法、使用經轉型之樹突狀細胞株DC2.4(已證明為APC之良好模型)[Mendoza L.等人,Prophylactic, therapeutic and anti-metastatic effects of BMDC and DC lines in mice carrying HPV 16-associated tumours
, Int J Oncol 23:243-7, (2003); Okada N.等人,Effects of lip ofectin-antigen complexes on major histocoMpatibility complex class I-restricted antigen presentation pathway in murine dendriticcells and on dendritic cell maturation
, Biochim Biophys Acta 1527:97-101, (2001)],研究DOTAP所誘導的全面基因調節(圖15A)。資料表明,DOTAP誘導數種趨化因子之過度表現,包括單核細胞化學引誘劑蛋白-1("MCP-1")/CC趨化因子-2("CCL2")、巨噬細胞炎性蛋白-1α("MIP-1α")/CC趨化因子-3("CCL3")、巨噬細胞炎性蛋白-1β(MIP-1β)/CC趨化因子-4("CCL4")。除誘導CC趨化因子外,由於在50 μMDOTAP處理16小時後,IL-1β減少且IL-1受體拮抗劑("IL1RA")增加,因此IL-1信號被下調。有趣的是,樹突狀細胞標記CD11c受DOTAP脂質體誘導,說明陽離子性脂質在樹突狀細胞成熟中起作用。使用鼠BMDC、藉由RT-PCR證明趨化因子mRNA之誘導(圖15B)。
如上文所提及,趨化因子參與淋巴細胞遷移並在免疫反
應中扮演重要角色。如上文所提及,趨化因子誘導可能說明陽離子性脂質體之主要佐劑活性。此假設有助於吾人進一步研究該過程潛在之詳細機制。使用由不同脂質製成之脂質體確定趨化因子之誘導是否為脂質體之普通現象。使用BMDC的結果表明,僅四級陽離子性脂質體(DOTAP及DOEPC)誘導MCP-1/CCL2自BMDC中釋放。中性脂質體(DOPC及DOG)及帶負電荷之脂質體(DOPS及DOPG)卻不能(圖16)。此外,DOTAP之三級胺類似物DODAP亦不誘導活性,說明該活性需要脂質中具有四級胺基頭基團。實驗中亦包括LPS作為陽性對照。圖16亦表明,由於48小時培育不形成較高水準之趨化因子產生,因此陽離子性脂質體所誘導之CCL2在24小時內達到最大量。吾人資料亦表明,DOTAP誘導之CCL2表現具有劑量依賴性(圖17)。5 μM DOTAP誘導BMDC產生大量的CCL2且在45-75 μM下達到最大誘導量。為鑑別BMDC中何種途徑涉及DOTAP誘導CCL2,使用特異於不同信號傳導途徑之若干種抑制劑。
PD98059("PD")及U-0126("U")(特異於ERK途徑之抑制劑)幾乎完全消除CCL2產生,但驚人地,SB203586("SB")(p38途徑之抑制劑)卻協同性地使DOTAP誘導CCL2增強(圖18)。吾人資料亦清楚地表明,由於PKC抑制劑GF109203X("GF")及Src激酶抑制劑PP2不具有抑制效應,因此PKC途徑及Src激酶不涉及DOTAP誘導CCL2產生。圖18亦表明,由於渥曼青黴素("Wort")及百日咳毒素("PTx")具有某些抑制效應,因此在DOTAP處理後,PI-3激酶及Gi依賴性G蛋
白質偶聯受體(GPCR)可能涉及CCL2釋放。
已知某些趨化因子受ERK途徑調節[Yoo J. K.等人,IL-18 induces monocyte chemotactic protein-1 production in macrophages through the phosphatidylinositol 3-kinase/Akt and MEK/ERK1/2 pathways
, J Immunol 175:8280-6, (2005)],且已在吾人系統中得到證實。吾人使用BMDC研究ERK途徑之活化的資料表明,在暴露10分鐘內,DOTAP誘導ERK磷酸化,且磷酸化ERK(pERK)係以高量保持至少40分鐘(圖19A)。此外,p38亦被DOTAP略微活化。然而,DOTAP培育之後,未偵測到IκB磷酸化及隨後降解(資料未展示),說明NF-κB不涉及DOTAP引發之信號轉導,與吾人上述發現相符[Cui Z.等人,Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier
, Mol Pharm 2:22-8, (2005)]。西方墨點法表明,p38之抑制亦可能協同性地增強DOTAP激發之ERK磷酸化,說明ERK途徑及p38可互逆調節(圖19B)。換言之,p38之活化可能抑制ERK之活化。吾人資料與圖18中之ELISA資料相符亦表明,由於渥曼青黴素抑制DOTAP誘導之ERK磷酸化,因此DOTAP誘導之ERK活化係由PI-3激酶所介導。然而,PTx(Gi抑制劑)對DOTAP誘導之ERK磷酸化僅展現微小的抑制效應(圖19C)。另一方面,由於GF及PP2對DOTAP誘導之ERK磷酸化無影響,因此PKC途徑及Src激酶不涉及此過程。
為進一步證實ERK途徑涉及由DOTAP引發的CCL2釋放,使用RNA干擾方法阻斷BMDC中之ERK基因表現。圖
20A指示,24小時處理之後,ERK1被特異性siRNA下調。
在經siRNA處理之細胞中,DOTAP誘導的CCL2釋放亦衰減,從而阻斷ERK1基因表現(圖20B)。然而,即使ERK1被下調,LPS亦持續誘導CCL2釋放(圖20B)。資料亦證明ERK1 siRNA處理不影響其他信號傳導途徑。
至此,資料證明活體外DOTAP誘導之ERK活化及CCL2之下游誘導(downstream induction)。吾人已調查以上信號傳導機制是否在活體外DOTAP之佐劑活性中起作用。使用DOTAP/E7脂質體疫苗調配物、以皮下方式將小鼠免疫,且收集引流淋巴結並藉由ELISA檢定CCL2。資料表明,免疫之後,CCL2積聚於引流淋巴結中,且在注射後第2天達到最大積累(圖21A)。該等資料係藉由免疫組織化學染色法驗證(圖21B)。該等資料亦證明,在DOTAP/E7處理後,更多淋巴細胞遷移至引流淋巴結,引起引流淋巴結擴大。
ERK及p38途徑之抑制劑亦調配於DOTAP/E7脂質體複合物中。DOTAP/E7/U-0126及DOTAP/E7/SB203580形成透明懸浮液,但PD98059難以調配於DOTAP/E7複合物中。穩定的DOTAP/E7/藥物調配物具有與DOTAP/E7調配物類似的ζ電位、粒徑及抗原負荷能力,說明藥物合併並未明顯改變疫苗之物理特性。對小鼠皮下注射DOTAP/E7/U-0126及DOTAP/E7/SB203580。與活體外資料相符,DOTAP/E7調配物誘導CCL2積累於引流淋巴結中且U-0126及SB-203580互逆地調節CCL2誘導(圖22A)。為判定ERK及p38途徑是否與DOTAP/E7之抗腫瘤活性相關,吾人已檢查
DOTAP/E7/藥物調配物之抗腫瘤活性。如圖22B中所示,經脂質劑量為100奈莫耳之DOTAP/E7治療的有腫瘤小鼠對TC-1腫瘤之生長呈現強烈的生長抑制效應。ERK抑制劑U-0126當以相似脂質劑量與DOTAP/E7共調配時,使DOTAP/E7之抗腫瘤活性完全消除。類似地,當小鼠接受與SB-203580共調配之DOTAP/E7之治療時,展現抗腫瘤活性之部分抑制,說明p38 ERK兩種途徑在DOTAP/E7複合物之抗腫瘤活性中扮演重要角色。
脂質體為由雙層水合兩性脂質組成的封閉微脂粒結構[Small D.M.,Surface and bulk interactions of lipids and water with a classification of biologically active lipids based on these interactions,
Federation Proc. 29:1320-1326, (1970)]。自1974年其被鑑別為佐劑以來[Allison A.G.及Gregoriadis G.,Liposomes as immunological adjuvants,
Nature 252: 252, (1974)],已對脂質體作為蛋白質及DNA疫苗之傳遞系統進行廣泛探究[Chen W.C.及Huang L.,Non-viral vector as vassine carrier,
Adv Genet 54:315-37, (2005); Gregoriadis G.等人,Vaccine entrapment in liposomes,
Methods 19:156-62, (1999); Perrie Y.等人,Liposome-mediated DNA immunization via the subcutaneous route,
J Drug Target 11:555-63, (2003)]。用於傳遞抗原的脂質體展現若干獨特優點。首先,在脂質組成、尺寸、層狀、電荷及製備方法上存在明顯的變通性,可針對特定應
用對其加以選擇。此外,考量載劑與抗原比率時,具有封裝或穩定結合之形式的脂質體微脂粒系統具有高效的抗原負荷。就安全角度而言,脂質體具有生物可降解性、生物相容性且具有低免疫原性[Copland M.J.等人,Lipid bases particulate formulations for the delivery of antigen,
Immunol Cell Biol 83:97-105, (2005); O'Hagan D.T.及Singh M.,Microparticles as vaccine adjuvants and delivery systems
, Expert Rev Vaccines 2:269-83, (2003)]。此外,脂質體亦已用於將蛋白質抗原導入細胞質內(cytosolic)I類MHC途徑中以產生CD8+
T細胞反應[Chikh G.及Schutze-Redelmeier M.P.,Liposomal delivery of CTL epitopes to dendritic cells
, Biosci Rep 22:339-53, (2002)]。
由於陽離子性脂質體可與帶負電荷之細胞膜相互作用,因此其廣泛用於基因治療及疫苗傳遞[Anderson P.,Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins
, Infect Immun 62:2536-44, (1994); Hasegawa A.等人,Nasal immunization with diphtheria toxoid conjugated-CD52 core peptide induced specific antibody production in genital tract of female mice
, Am J Reprod Immunol 34:305-11, (2002)]。然而,就能夠活化先天免疫反應而言,陽離子性脂質體本身已被認為呈惰性[Moinegeon P.等人,Towards the rational design of Thl adjuvants,
Vaccine 19:4363-72, (2001)]。然而,陽離子性
脂質體亦可誘導共刺激性分子CD80及CD83之表現且活化人類樹突狀細胞(圖23A-23E及25A-25B)。其亦誘導人類樹突狀細胞產生涉及針對疾病形成強免疫反應的關鍵趨化因子及細胞因子(圖24A-24F)。該等發現皆說明,陽離子性脂質體除有效傳遞至樹突狀細胞外,亦為免疫系統之強活化劑。
與目前所用的佐劑(諸如Alum、CpG及CFA)不同,陽離子性脂質免疫調節劑不增強NF-κB之表現,證明陽離子性脂質刺激免疫系統係經由NF-κB無關機制進行信號傳導,從而將吾人基於陽離子性脂質之療法中類似LPS之炎性反應之機率排除。該等研究表明,陽離子性脂質體除為有效傳遞系統外,亦屬於具有改良之安全分布的獨特類免疫刺激劑。
在本說明書中,對陽離子性脂質體之佐劑活性之可能分子機制加以研究。調查MAP激酶信號傳導途徑之各種組分在陽離子性脂質之免疫刺激活性中的作用。脂質經調查為樹突狀細胞之活性刺激劑(active stimulator)。證明趨化因子產生係由陽離子性脂質誘導且該誘導主要係由ERK途徑介導。此外,資料明顯表明p38途徑對該過程負調節。最新報導證明,p38在IL-2產生中起負面作用,主要經由其調節ERK之活性之能力介導。p38特異性抑制劑誘導ERK活化,最終導致IL-2基因活化增強[Kogkopoulou O.等人,Conditional up-regulation of IL-2 production by p38 MAPK inactivation is mediated by increased Erkl/2 activity
, J
Leukoc Biol 79:1052-60, (2006)]。p38之磷酸化及活化使其與ERK1/2的相互作用增強且與ERK1/2磷酸轉移酶活性之抑制相關,說明活化之p38可能隔離ERK1/2並空間阻斷MEK1將其磷酸化[Zhang H.等人,Stress-reduced inhibition of ERKl and ERK2 by direct interaction with p38 MAP kinase
, J Biol Chem 276:6905-8, (2001)]。最新研究說明,ERK之穩定且持續之磷酸化導致樹突狀細胞中AP-1轉錄因子c-Fos之磷酸化,此又抑制Th
1限定細胞因子IL-12之表現,從而有利於Th
2偏移。然而,吾人結果展現與彼等報導的兩大差異。首先,彼等研究中所用之Pam-3-cys(TLR-2配位體)展現比LPS更強的ERK活化(Dillon等人,2004),而陽離子性脂質誘導之ERK活化小於所觀測之LPS。吾人亦檢測了Th
2細胞因子IL-10,且結果表明DOTAP不誘導IL-10(資料未展示)。因此,DOTAP誘導ERK之信號不足以激發Th
2反應。其次,DOTAP誘導之IL-1信號之下調亦可能抑制Th
2反應。DOTAP誘導之ERK活化似乎得到適當地控制。
吾人結果說明DOTAP誘導ERK活化及誘導CCL2需要PI-3激酶。鑑定PI-3激酶與ERK之間的確切關係需要進一步的調查。儘管ELISA資料表明PTx抑制DOTAP誘導CCL2釋放,但如西方墨點法資料所示,DOTAP誘導之ERK磷酸化僅略微地受PTx抑制。因此,G蛋白質偶聯受體可能涉及或可能不涉及由DOTAP引發的信號轉導。有趣地是,溶血磷脂醯膽鹼(LPC)誘導G2A受體依賴性ERK活化及T細胞遷
移[Radu C.G.等人,T cell chemotaxis to lysophosphatidylcholine through the G2A receptor,
Proc Natl Acad Sci U S A 101:245-50, (2004)]。著者最初報導LPC為G2A受體之配位體[Kabarowski J.H.等人,Lysophosphatidylcholine as a ligand for the immunoregulatory receptor G2A
, Science 293:702-5, (2001)],但隨後證明LPC誘導之ERK活化係經由G2A受體之細胞內隔離(intracellular sequestration)及表面表現之調節機制進行[Wang L等人,Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G protein-coupled receptor G2A
, Mol Biol Cell 16:2234-47, (2005)]。活性陽離子性脂質含有一個大極性頭基團及兩個長醯基鏈。該等結構特性可有利於其插入脂質膜內,產生脂質單層之自生彎曲部分之可能變化以及膜蛋白質之構形及功能變化。
由於p38負調節CCL2誘導之ERK活化,因此吾人預期調配於DOTAP/E7疫苗中的p38抑制劑SB203580可能增強抗腫瘤活性。然而,卻發現相反結果,意味著p38亦可能在DOTAP/E7誘導之腫瘤消退中扮演積極角色。由於陽離子性脂質體可誘導活性氧(ROS)之形成,從而引起p38活化,因此DOTAP誘導之p38活化可能經由ROS進行(Iwaoka等人,2006)。大量證據表明,p38活化誘導Th
1細胞因子自樹突狀細胞中釋放[DeSilva D.R.等人,The p38 mitogen-activated protein kinase pathway in activated and Anergic Thl cells
, Cell Immunol 180:116-23, (1997); Yu J.J.等人,Regulation and phenotype of an innate Thl cell: role of cytokines and the p38 kinase pathway,
J Immunol 171:6112-8, (2003)]。在哺乳動物種類中,MAP激酶(ERK、p38及JNK)涉及免疫反應之所有方面(從先天性免疫之起始期至適應性免疫之活化,及至免疫功能結束時的細胞死亡)[Dong C.等人,MAP kinases in the immune response,
Annu Rev Immunol 20:55-72, (2002)]。
許多種實驗性佐劑可供用於動物且其中一些佐劑已經歷臨床試驗測試。其包括幾種油包水乳液、脂質體及其他化學佐劑[Vogel, F. R.及Powell,
M. F.A compendium of vaccine adjuvants and excipients.
Pharm Biotechnol 6:141 (1995)]。然而,除鋁鹽外,僅流感病毒體[Gluck, R.及Walti, E.,Biophysical validation of Epaxal Berna, a hepatitis A vaccine adjuvanted with immunopotentiating reconstituted influenza virosomes (IRIV).
Dev Biol (Basel) 103:189 (2000)]及Chiron之MF59 [Kahn, J. O.等人,Clinical and immunologic responses to human immunodeficiency virus (HIV) type 1SF2 gp120 subunit vaccine combined with MF59 adjuvant with or without muramyl tripeptide dipalmitoyl phosphatidylethanolamine in non-HlV-infected human volunteers
. J Infect Dis 170:1288 (1994)]已投入市場。類似於陽離子性脂質體(未公開資料),基於亞微乳液之佐劑MF59被樹突狀細胞內在化[Dupuis, M.等人,Dendritic cells internalize vaccine adjuvant after intramuscular injection.
Cell Immunol 186:18 (1998)]。其刺激多種免疫活性,產生針對共傳遞之抗原的高抗體及T-細胞反應。然而,根據有關HSV及流感疫苗的臨床試驗報導[Jones, C. A.及Cunningham, A. L.,Vaccination strategies to prevent genital herpes and neonatal herpes simplex virus (HSV) disease.
Herpes 11:12 (2004);及Minutello, M.等人,Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons
. Vaccine 17:99 (1999)],獲自動物模型之證據說明,MF59佐劑使中和抗體反應而非T-細胞反應增強。因此,作為疫苗佐劑的陽離子性脂質體與MF59不同之處在於其形成如吾人資料所示的強細胞介導免疫反應。
曾經報導陽離子性脂質/抗原複合物似乎為最簡單的癌症疫苗調配物。其僅含有兩個分子,亦即抗原及載劑。除將E7肽傳遞至抗原呈現細胞(諸如樹突狀細胞)之細胞質外,DOTAP亦必須活化DC。實際上,僅由DOTAP組成的脂質體誘導樹突狀細胞中CD80/CD86共刺激性分子之表現。
總之,吾人之發現首次說明陽離子性脂質體為免疫系統強刺激劑。本文中所報導之結果闡明了諸如DOTAP之陽離子性脂質之佐劑活性之分子機制。
DOTAP介導幾種趨化因子及細胞因子(其表現由ERK途
徑介導)之誘導。吾人之研究亦鑑定ERK途徑為用於評價陽離子性脂質體之佐劑活性的新穎分子標記。該等標記可用於高產量篩檢或設計基於脂質之強佐劑及疫苗傳遞系統。
證明陽離子性脂質/抗原複合物對人類免疫系統之細胞的免疫刺激能力
如上所述製備陽離子性脂質體。用於調配物中的E7抗原為藉由HLA-A*0201(HPV-16 E7,胺基酸11-20,YMLDLQPETT(SEQ. ID. NO. 2))限制的已鑑別人類E7肽。該肽係由University of Pittsburgh (Molecular Medicine Institute, Pittsburgh, PA)合成。人類HLA-A2人類樹突狀細胞獲自Lonza (Walkersville, MD)。將經冷凍之凍存管(cryovial)解凍,且在37℃及5%CO2
下,將樹突狀細胞於12孔組織培養皿中於2 ml培養基中以125,000個細胞/平方公分之初始塗覆密度培養於補充有50微克/毫升IL-4及GM-CSF的LGM-3培養基(購自MD Walkersville之Lonza)中。將細胞在培養液中生長3天,且經顯微鏡檢查,呈現為黏附性及圓形細胞之混合物。
在第3日,用50微克/毫升劑量之新配IL-4及GM-CSF處理細胞(所有孔),且測試孔用10 ng/mL之介白素1-β("IL-β")、介白素6("IL-6")及TNF-α與10 μg/ml之前列腺素
E2("PGE-2")之混合物處理(活化之陽性對照);不處理(活化之陰性對照);及用最終濃度為2.5、10及40微莫耳濃度的DOTAP/E7處理;用最終濃度為2.5、10及40微莫耳濃度的DOEPC處理;及用脂質及膽固醇之最終濃度為2.5、10及40微莫耳濃度的DOTAP/膽固醇/E7處理。添加25 mol%之膽固醇(脂雙層之穩定劑)。將經處理之樹突狀細胞在培養液中維持24小時且收穫供細胞表面標記染色及流式細胞術分析之用。藉由血球計將所收穫之細胞計數,且將10 μl之以下抗體結合物依序添加至各樣本中用於標示表面標記:CD80-FITC、CD83-APC及CD86-PE(BD Biosciences)。
隨後使用BD FACxcaliber流式細胞儀藉由流式細胞術分析表面經標記之細胞,且監測活化後所產生的共刺激性樹突狀細胞標記分子CD80、CD83及CD86。如圖23A、23B及23C中所示,經具有不同脂質劑量之陽離子性脂質/E7複合物處理的初始人類樹突狀細胞使所評價的且為將抗原成功呈現給T-細胞所需的樹突狀細胞活化之全部三種共刺激性標記之表現上調。如圖23D及23E中所示,醫藥組合物中亦可包括諸如穩定劑之其他組分而對複合物之免疫刺激能力無負面影響。
如上所述處理人類HLA-A2樹突狀細胞(Lonza, Walkersville, MD)並生長於培養液中。在第3日,將細胞用40微莫耳濃度之DOTAP/E7複合物或50微莫耳濃度之強免
疫刺激劑脂多醣(LPS)(陽性對照)處理。移除檢定孔中的培養基且於微型離心機中以1300 rpm離心5分鐘以使未附著之樹突狀細胞成團。移除上層清液並以每毫升10微升之Calbiochem (La Jolla, CA)蛋白酶抑制劑混合液組I(目錄號第539131號)處理並在分析之前冷凍儲存。藉由Pierce Biotechnology (Woburn, MA)、使用其Searchlight蛋白質陣列多路ELISA檢定法分析樣本的細胞因子表現。
評價TNF-α及IL-12之產生,並對已知為細胞免疫反應中所必需之選定趨化因子CCL3、CCL4、CCL5及CCL19之產生進行全面評價(圖24A-24F)。圖24說明,DOTAP/E7複合物誘導人類樹突狀細胞產生細胞因子及趨化因子。圖24A-24F說明DOTAP/E7分別誘導TNF-α、IL-12、CCL3、CCL4、CC15及CCL-19之產生。
類似於對鼠樹突狀細胞所作之觀測(實例1),與LPS不同,DOTAP/E7複合物不誘導促炎性細胞因子TNF-α之明顯產生,此證明在人類系統中,NF-κB介導之信號傳導亦不被陽離子性脂質活化。DOTAP/E7刺激多種趨化因子之有效產生證明,陽離子性脂質起強免疫刺激劑之作用,且為鼠免疫細胞與人類免疫細胞之有效活化提供類似的相關性。
如上所述處理人類HLA-A2樹突狀細胞(Lonza, Walkersville, MD)並生長於培養液中。在第3天,將測試孔用10 ng/mL之IL-β、IL-6及TNF-α與10 μg/ml之PGE-2之混
合物處理(活化之陽性對照);不處理(活化之陰性對照);用50微莫耳濃度之強免疫刺激劑脂多醣(LPS)處理(第二個陽性對照);或用粒徑為100 nm、200 nm及400 nm之10微莫耳濃度DOTAP/E7複合物處理。圖25中所示的結果證明,在免疫療法之開發中,陽離子性脂質/抗原複合物可在誘導免疫反應的寬粒徑範圍內使用。
實例1及實例2中所述的研究可用於開發由陽離子性脂質/抗原複合物組成的免疫治療調配物。經諸如DOTAP及DOEPC之陽離子性脂質刺激後,鼠骨髓源樹突狀細胞(BMDC)被活化以便表現共刺激性分子CD80及CD86[Vangesseri等人,Immunostimulation of dendritic cells by cationic liposomes
. Mol Membr Biol 23:385-395 (2006)]。
在DOTAP活化之BMDC中,亦觀測到MAP激酶信號傳導途徑之各種組分及幾種趨化因子(諸如CCL2)之活化。動物研究說明,DOTAP脂質體在特定劑量範圍內充當抗原載劑與強佐劑以便誘導活化之樹突狀細胞遷移至引流淋巴結,從而引起活體內產生針對具有抗原之細胞(諸如腫瘤細胞)的抗原特異性CD8+
T淋巴細胞。對初始人類樹突狀細胞之活化的活體外測試表明,陽離子性脂質為強免疫刺激劑,促進樹突狀細胞活化,進而表現T-細胞識別及抗原呈現所需的共刺激性分子。吾人亦已證明,人類樹突狀細胞在被DOTAP/E7脂質體活化後,促進人類T-細胞活體外明顯增殖。
以上所報導之研究鑑別了陽離子性脂質體之具體獨特組成及應用,其可用於開發針對若干種衰竭性疾病的簡單、成本有效且迫切需要的免疫療法。
由於可對上述態樣及例示性實施例作出不同更改而不背離本發明之範圍,因此以上說明中所含的全部內容應解釋為具有說明性而非具有限定性。為此,儘管該等實例主要論述陽離子性脂質DOTAP、DOEPC及DOTMA,但熟習此項技術者應瞭解該等陽離子性脂質僅具有例示性且該等方法可應用於其他陽離子性脂質。
圖1圖示用1,2-二油醯基-3-三甲基銨丙烷("DOTAP")刺激樹突狀細胞之後,活體外細胞因子之產生情況。
圖2展示藉由流式細胞術對CTL介導細胞毒性的分析。
製備並擴展E7-特異性細胞毒性T-淋巴細胞("CTL")純系。
用E7肽活體外再刺激五天後,將效應細胞與經PKH-67標記之TC-1靶細胞一起、以指定效應細胞:靶細胞比率、在37℃下培育4小時。BL-6用作非特異性靶細胞對照。E7-特異性殺死百分比係以流式細胞術中選通PKH-67陽性靶細胞內之PI陽性細胞之百分比測定。在100:1 E:T比率下,藉由與對照組比較來對統計分析進行計算(*p<0.01,**p<0.001,n=5)。
圖3展示對以最佳脂質劑量接受DOTAP/E7的小鼠觀測腫瘤浸潤性T-淋巴細胞。TC-1腫瘤係如所述建立且保持不治療或在第6日單次注射治療。第14日解剖實體腫瘤並檢測
浸潤性淋巴細胞。使用FITC結合之抗-CD8(A、B、C)及抗-CD4(D、E、F)抗體測定腫瘤浸潤性T細胞,繼之用DAPI對比染色。如所述檢查來自3隻小鼠組的代表性腫瘤切片且用共焦顯微術成像。執行TUNEL檢定以偵測腫瘤切片之細胞凋亡(G、H、I)。
圖4圖示藉由用陽離子性脂質治療法治療而誘導體液活性的情況。
圖5圖示說明使用流式細胞術,小鼠在接受具有E7肽之DOTAP之後,調節性T細胞群減少。
圖6展示經DOTAP/E7調配物治療之小鼠中之TC-1腫瘤生長動力學。TC-1接種後第6日,小鼠接受10 μg以不同脂質濃度調配於DOTAP脂質體中之E7肽之治療。將第23日各組之TC-1腫瘤尺寸與未經治療之對照組對比且進行統計分析(*p<0.05,**p<0.01,***p<0.001)。
圖7展示皮下注射DOTAP誘導樹突狀細胞活化並遷移至引流淋巴結。對未處理小鼠(n=4-6)注射PBS對照劑(A、C及E)或含有100 nmol總脂質、具有0.5% NBD-DOTAP的DOTAP/E7 (B、E及F)。在注射後第4小時,製備引流淋巴結細胞並用針對表面標記之適當抗體染色。對全部淋巴結細胞內NBD與CD11c(A及B)或CD86(C及D)之共表現進行分析,而對CD3+
群內之NBD與CD8之共表現(E及F)進行選通並分析。數字表示象限內細胞之百分比。
圖8展示以調配於最佳DOTAP佐劑中的E7肽進行免疫誘發功能性CD8+
T細胞。每105
個CD8+
T細胞之CD8+
IFN-γ+
細胞的數目係以平均值±SD展示且與未經治療之對照組對比(n=4,**p<0.01)。
圖9圖示說明與已知可誘發T-細胞反應之強佐劑相比,由不同陽離子性脂質組成之陽離子性脂質/E7複合物的抗腫瘤免疫反應。
圖10圖示說明小鼠引流淋巴結中之ROS產生情況且ROS產生與抗腫瘤免疫反應相關。A:注射之後第2小時將來自已接受含有0、15、100或600 nmol脂質之DOTAP/E7之注射的小鼠引流淋巴結("DLN")分離。列出具有陽性ROS信號之細胞的相對百分比。B:DLN中之細胞毒性係藉由在注射DOTAP/E7之後第10小時收集細胞、藉由流式細胞術量測。展示樹突狀細胞(DC)中死細胞(PI陽性)之相對百分比(空心方塊)及每105
個淋巴結(LN)細胞之活DC數且藉由配對學生t檢驗與未經治療之對照組比較(**P<0.01)。C:藉由將惰性中性脂質二油醯基磷脂醯膽鹼("DOPC")與DOTAP/E7共調配(DOPC/DOTAP之莫耳比=5)使DLN中之活性氧("ROS")產生減少,導致複合物之抗腫瘤活性減小。
圖11說明改良之封裝效率對由陽離子性脂質/抗原複合物所引起之免疫反應的影響。抗TC-1腫瘤活性係藉由將E7-脂肽併入陽離子性脂質調配物中得到增強。在第6日,具有TC-1腫瘤之小鼠(每組8-12隻小鼠)接受DOTAP/E7(含有5 nmol或10 nmol E7肽)或DOTAP/E7-脂肽(含有5 nmol肽)或單獨DOTAP脂質之單次治療。經右旋糖(5%)治療之
小鼠用作陰性對照。將第24日各組之腫瘤平均尺寸與接受DOTAP/E7(5 nmol)之組之腫瘤平均尺寸對比且藉由學生t-檢驗分析(*p<0.05,**p<0.01)。
圖12圖示證明用DOTAP/E7-脂肽使小鼠免疫誘導CD8+
T細胞中IFN-γ分泌之增強。
圖13圖示說明與不相關短胺基酸序列連接之肽抗原之免疫原性增強。
圖14A圖示說明經不同陽離子性脂質體刺激之後,共刺激性分子CD 80與CD 86在DC2.4細胞上之表現。
圖14B圖示說明關於共刺激性分子(CD 80)於DC2.4細胞上之表現的陽離子性脂質之烴鏈長度依賴性之表現。
圖15展示藉由陽離子性脂質體使趨化因子及CD11c之轉錄上調且使IL-1下調。(A)Affymetrix微陣列分析,展示DC2之後mRNA含量之倍數增加。將四種細胞用50 μMDOTAP脂質體處理24個小時。(B)RT-PCR,表明在經50 μM DOEPC或DOTAP脂質體培育16小時、繼之總RNA萃取且經特異性引子擴增之BMDC中,CCL2及CCL4被DOTAP及1,2-二油醯基-sn-甘油基-3-乙基磷酸膽鹼("DOEPC")上調(100 ng/mL LPS作為陽性對照)。
圖16展示僅陽離子性脂質體誘導CCL2自BMDC中釋放。
圖17展示DOTAP以劑量依賴性方式誘導CCL2釋放。
圖18展示使用MAP激酶抑制劑研究在DOTAP誘導CCL2自BMDC釋出中所涉及的信號傳導途徑。在第6日,將BMDC用抑制劑預培育20分鐘,繼之用75 μM DOTAP脂質
體處理24小時。實驗中所使用之濃度為:PD(PD-98059):20 μM;SB(SB-203580):10 μM;U(U-0126);10 μM;PTx(百日咳毒素):200 ng/mL;PP2:10 μM;Wort(渥曼青黴素):200 nM;GF(GF109203X):200 nM。藉由ELISA分析上層清液。100奈克/毫升之LPS充當陽性對照。
圖19展示DOTAP誘導BMDC中ERK及p38之活化。(A)時程研究(Time course study)。在第6日,將BMDC以106
個/毫升/孔之密度接種於12孔板中。將其用75 μm DOTAP脂質體培育指定時間。收穫細胞並使用圖式中所指定之抗體進行西方墨點分析。用作為負載對照之ERK2抗體探測相同膜。(B)DOTAP誘導之ERK活化被p38負調節。(C)DOTAP誘導之ERK活化主要經由PI-3激酶進行且被p38負調節。
圖20展示藉由siRNA方法下調ERK基因表現使DOTAP誘導之CCL2自BMDC中釋放減弱。(A)在BMDC中處理24小時之後,ERK1基因表現被siRNA阻斷。(B)siRNA阻斷ERK1特定地使DOTAP誘導之CCL2自BMDC中釋放減弱。DOTAP:75_M,LPS:100 ng/mL。*p
<0.05(與對照siRNA相比),n
=3。
圖21展示DOTAP/E7調配物誘導CCL2積累於小鼠引流淋巴結中。在第0日,對小鼠(n=3)注射DOTAP/E7調配物(100 nmol之DOTAP及10 μg之E7肽)。在指定日期,處死小鼠且收集引流淋巴結。將引流淋巴結勻化於100 μl ELISA緩衝液(10% FBS於PBS中)且接著如正文中所提及,(A)藉由
ELISA分析或(B)藉由CCL2抗體、以免疫化學方式染色。原放大倍數:400倍。
圖22展示抑制ERK使引流淋巴結中CCL2積累減少且阻斷DOTAP/E7調配物之抗腫瘤活性。(A)CCL2於引流淋巴結中之積累係藉由ERK及p38途徑互逆調節。(B)在接受與或不與抑制劑共調配之DOTAP/E7的小鼠中之TC-1腫瘤生長動力學。在第0日將TC-1細胞(1×105
個/小鼠)注入小鼠(n=5)。在第6日,用DOTAP/E7、DOTAP/E7/U-0126、DOTAP/E7/SB或PBS治療小鼠。爾後量測腫瘤尺寸。與DOTAP/E7相比,*p
<0.05。
圖23展示陽離子性脂質/抗原複合物有效地活化人類樹突狀細胞。圖23A-23E分別說明藉由DOTAP/E7刺激CD80、藉由DOTAP/E7刺激CD 83、藉由DOTAP/E7刺激CD86、藉由DOEPC/E7及DOTAP/膽固醇/E7刺激CD 80以及藉由DOEPC/E7及DOTAP/膽固醇/E7刺激CD 83之情況。
圖24說明DOTAP/E7複合物誘導人類樹突狀細胞產生細胞因子及趨化因子之能力。圖24A-24F分別說明TNF-α、IL-12、CCL3、CCL4、CC15及CCL-19之產生。
圖25說明陽離子性脂質/抗原複合物粒徑對人類樹突狀細胞之活化的影響。
<110>美商PDS生技公司<120>利用陽離子性脂質刺激免疫反應<130>PDSB-08 <140>097110452 <141>2008-03-24 <150>60896412 <151>2007-03-22 <150>60911549 <151>2007-04-13 <150>60948512 <151>2007-07-09 <150>60983799 <151>2007-10-30 <160>9 <170>PatentIn version 3.5 <210>1 <211>9 <212>PRT <213>16型人類乳頭狀瘤病毒<400>1
<210>2 <211>10 <213>16型人類乳頭狀瘤病毒
<400>2
<210>3 <211>20 <212>DNA <213>C57BL/6小鼠<400>3
<210>4 <211>21 <212>DNA <213>C57BL/6小鼠<400>4
<210>5 <211>20 <212>DNA <213>C57BL/6小鼠<400>5
<210>6 <211>20 <212>DNA <213>C57BL/6小鼠<400>6
<210>7 <211>20 <212>DNA <213>C57BL/6小鼠<400>7
<210>8 <211>22 <212>DNA <213>C57BL/6小鼠<400>8
<210>9 <211>22 <212>DNA <213>C57BL/6小鼠<400>9
(無元件符號說明)
Claims (35)
- 一種包含至少一種陽離子性脂質之組合物,該陽離子性脂質之劑量足以活化個體免疫系統細胞之有絲分裂原活化蛋白質("MAP")激酶信號傳導途徑來誘導該個體之免疫刺激佐劑效應,該效應包括T-細胞或抗體或其組合;其中陽離子性脂質之小鼠劑量為介於10nmol及約400nmol間;且其中陽離子性脂質之哺乳動物劑量係由該小鼠劑量測定。
- 一種包含至少一種陽離子性脂質之組合物,該陽離子性脂質之劑量足以降低個體之調節性T細胞群,其中陽離子性脂質之小鼠劑量為介於30nmol及約400nmol間,且其中陽離子性脂質之哺乳動物劑量係由該小鼠劑量測定。
- 如請求項1之組合物,其中該陽離子性脂質係藉由刺激細胞外信號調節激酶("ERK")1、ERK 2及p38中之至少一者來活化MAP激酶信號傳導途徑。
- 如請求項1至3中任一項之組合物,其中該陽離子性脂質包含具有下式所示之結構的非類固醇陽離子性脂質:
- 如請求項1至3中任一項之組合物,其中該陽離子性脂質係選自DOTAP、DOTMA、DOEPC及其組合。
- 如請求項1至3中任一項之組合物,其中該陽離子性脂質係DOTAP。
- 如請求項1至3中任一項之組合物,其進一步包含至少一種抗原以形成陽離子性脂質/抗原複合物,其中該陽離子性脂質/抗原複合物刺激抗原特異性免疫反應。
- 如請求項7之組合物,其中該至少一種抗原係選自腫瘤相關抗原、病毒抗原、微生物抗原及其組合。
- 如請求項8之組合物,其中該至少一種抗原包括脂質化抗原或經修飾以增強疏水性之抗原,且該抗原與所連疏水基之間視需要包含連接子。
- 如請求項9之組合物,其中該脂質化抗原係與棕櫚酸脂質化。
- 如請求項10之組合物,其中該棕櫚酸係藉由間隔子與該抗原連結,該間隔子之胺基酸序列為K-S-S。
- 如請求項7之組合物,其中該至少一種抗原係選自脂蛋白、脂肽、經具有強疏水性之胺基酸序列修飾的蛋白質或肽,及其組合。
- 一種包含至少一種陽離子性脂質之組合物於製備治療病患疾病或感染之藥物之用途,其中該陽離子性脂質之劑量足以活化該病患之MAP激酶信號傳導途徑來誘導免疫刺激佐劑效應; 其中陽離子性脂質之小鼠劑量為介於10nmol及約400nmol間;且其中陽離子性脂質之哺乳動物劑量係由該小鼠劑量測定。
- 一種包含至少一種陽離子性脂質之組合物於製備誘導免疫系統中免疫刺激佐劑效應之藥物之用途,其中該陽離子性脂質之劑量足以活化MAP激酶信號傳導途徑來誘導免疫刺激佐劑效應;其中陽離子性脂質之小鼠劑量為介於10nmol及約400nmol間;且其中陽離子性脂質之哺乳動物劑量係由該小鼠劑量測定。
- 一種包含至少一種陽離子性脂質之組合物於製備降低有需要病患中之調節性T細胞群之藥物之用途,其中該陽離子性脂質之劑量足以降低該病患之調節性T細胞群;其中陽離子性脂質之小鼠劑量為介於10nmol及約400nmol間;且其中陽離子性脂質之哺乳動物劑量係由該小鼠劑量測定。
- 如請求項13或14之用途,其中該陽離子性脂質係藉由刺激ERK 1、ERK 2及p38中之至少一者來活化MAP激酶信號傳導途徑。
- 如請求項13至15中任一項之用途,其中該陽離子性脂質包含具有下式所示之結構的非類固醇陽離子性脂質:
- 如請求項13至15中任一項之用途,其中該陽離子性脂質係選自DOTAP、DOTMA、DOEPC及其組合。
- 如請求項13至15中任一項之用途,其中該陽離子性脂質係DOTAP。
- 如請求項13至15中任一項之用途,其中該組合物進一步包含至少一種抗原以形成陽離子性脂質/抗原複合物,其中該陽離子性脂質/抗原複合物刺激抗原特異性免疫反應。
- 如請求項20之用途,其中該至少一種抗原係選自腫瘤相關抗原、病毒抗原、微生物抗原及其組合。
- 如請求項20之用途,其中該至少一種抗原包括脂質化抗原或經修飾以增強疏水性之抗原,且該抗原與所連疏水基之間視需要包含連接子。
- 如請求項22之用途,其中該脂質化抗原係與棕櫚酸脂質化。
- 如請求項23之用途,其中該棕櫚酸係藉由間隔子與該抗原連結,該間隔子之胺基酸序列為K-S-S。
- 如請求項20之用途,其中該至少一種抗原係選自脂蛋白、脂肽、經具有強疏水性之胺基酸序列修飾的蛋白質 或肽,及其組合。
- 如請求項1之組合物,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於30nmol及400nmol間。
- 如請求項1之組合物,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於50nmol及400nmol間。
- 如請求項1之組合物,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於75nmol及400nmol間。
- 如請求項2之組合物,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於50nmol及400nmol間。
- 如請求項2之組合物,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於75nmol及400nmol間。
- 如請求項14之用途,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於30nmol及400nmol間。
- 如請求項14之用途,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於50nmol及400nmol間。
- 如請求項14之用途,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測 定,其中該小鼠劑量為介於75nmol及400nmol間。
- 如請求項15之用途,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於50nmol及400nmol間。
- 如請求項15之組合物,其中該至少一種陽離子性脂質之足以刺激哺乳動物中免疫反應之劑量係由該小鼠劑量測定,其中該小鼠劑量為介於75nmol及400nmol間。
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JP6211383B2 (ja) | 2017-10-11 |
EA200901270A1 (ru) | 2010-04-30 |
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CN101702882A (zh) | 2010-05-05 |
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US20090017057A1 (en) | 2009-01-15 |
JP2014058525A (ja) | 2014-04-03 |
WO2008116078A3 (en) | 2009-06-04 |
CN107670031A (zh) | 2018-02-09 |
TW200902060A (en) | 2009-01-16 |
WO2008116078A2 (en) | 2008-09-25 |
WO2008116078A4 (en) | 2009-10-15 |
AU2008228798A1 (en) | 2008-09-25 |
US20150093410A1 (en) | 2015-04-02 |
WO2008116078A9 (en) | 2008-11-20 |
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