CN102281892A - 酿酒酵母线粒体核酸级分用于免疫刺激的用途 - Google Patents
酿酒酵母线粒体核酸级分用于免疫刺激的用途 Download PDFInfo
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Abstract
本发明涉及酿酒酵母线粒体核酸级分和抗原用于制备使免疫应答朝向针对所述抗原的Th1型应答、更尤其地用于预防和/或治疗癌症、传染病和变态反应的药物组合物的用途。也提供了具有协同效应的佐剂组合物、具有协同效应的疫苗组合物和试剂盒。也提供了治疗个体的方法。
Description
技术领域
本发明一般涉及佐剂。具体而言,本发明涉及具有佐剂作用的酿酒酵母(Saccharomyces cerevisiae)线粒体核酸级分用于制备药物组合物的用途,其中所述的药物组合物旨在使免疫应答朝向抗特定抗原的Th1型应答。
发明背景
多年来,接种技术基本上一直是将抗原(例如蛋白质、死病毒或减毒病毒)引入动物以引起针对感染性生物的免疫应答。自从80年代末,新接种技术已经出现,其为将包含编码该抗原的核酸序列的载体导入动物中。例如,编码狂犬病病毒糖蛋白的活痘苗病毒已经成功地在西欧国家用于消除陆生动物狂犬病(CLIQUET等人,消除西欧国家陆生动物狂犬病(Elimination of terrestrial rabies in Western European countries).Developments in biologicals.2004,第119卷,第185-204页)。核酸免疫的主要优点是细胞免疫(包括CD4+和CD8+T细胞)和体液免疫应答均可以被诱导,这是因为编码的抗原可以不仅经过内源途径加工也经由外源途径被加工,肽表位通过I类主要组织相容性复合物(MHC)以及II类复合物呈递(HAUPT,等人.针对肿瘤相关抗原的DNA疫苗接种用于抗肿瘤疗法的潜力(The Potential of DNA Vaccination against Tumor-Associated Antigensfor Antitumor Therapy).Experimental Biology and Medicine.2002,第227卷,第227-237页)。
细胞毒性T淋巴细胞(CTL)应答的高效产生已经为通过核酸疫苗接种预防或治疗癌症铺平了道路。许多肿瘤细胞表达叫做TAA(肿瘤相关抗原)的特异性抗原,但是免疫系统(因肿瘤周围的因子而下调)对这些抗原的识别不良。用编码TAA的核酸疫苗接种患者,导致TAA在免疫系统完全有效的环境中表达,引起特异性针对该肿瘤细胞的免疫应答。
然而,尽管疫苗接种继续为迄今最成功的干预性健康策略,但传染病和癌症仍是世界范围死亡的主要原因。接种不能产生有效免疫的一个主要原因是缺少能够启动所希望免疫应答的合适佐剂。另外,大多数常规的佐剂是成分不太明确的复杂物质,不能满足新一代疫苗所期望的严格安全性和功效标准。
通过激活先天免疫起作用的新一代佐剂为开发更安全、更有效的疫苗提供了令人激动的机会。Toll样受体(TLR)家族似乎在先天免疫系统中扮演着检测病原体表达的高度保守分子的关键角色。为了能够快速检测感染,目前已知在人类中表达的10种TLR之每一种明显地已经进化为当如下某些类型的病原体表达分子存在时受到刺激,所述的病原体表达分子在宿主细胞中不表达,或被隔绝在TLR不可获得它们的细胞区室中。合适病原体分子对TLR的活化充当着启动适宜免疫防御的″报警信号″。这些TLR激活物也已经成功地单独用来加强对抗病原体或肿瘤细胞的天然免疫应答。例如,CpG寡脱氧核苷酸(ODNs)是TLR9激动剂,其作为疫苗佐剂以及在治疗癌症、感染、哮喘和变态反应方面显示出有希望的结果。开发了它们中之一,CPG-7909,作为单一疗法以及作为佐剂与化疗法和免疫疗法组合用于癌症的治疗。I期和II期试验已经在几种造血系统肿瘤和实体肿瘤中检查了这种药物(MURAD,等人.癌症疗法中的CPG-7909(PF-3512676,ProMune):toll样受体-9激动剂(CPG-7909(PF-3512676,ProMune):toll-like receptor-9 agonist in cancer therapy).Expert opinion on biologicaltherapy.2007,第7卷,第8期,第1257-1266页)。
免疫应答的性质反映了由免疫刺激的抗原特异性淋巴细胞谱(profile)。淋巴细胞、尤其T细胞,由亚群组成,这些亚群可以被不同类型抗原刺激并执行不同效应子功能。例如,在病毒感染中,病毒抗原在感染的细胞中合成并且在与I类MHC分子结合的情况下呈递,从而导致对CD8+I类MHC限制性CTL的刺激。相反,胞外微生物抗原被APC内吞、加工并且偏好地在与II类MHC分子结合的情况下呈递。这活化CD4+、II类MHC限制性辅助T细胞,导致抗体产生和巨噬细胞活化、但是相对无效的CTL形成。甚至在CD4+辅助T细胞群体内,也存在应答抗原性刺激而产生不同细胞因子的亚类。在初始遭遇抗原后,幼稚CD4+T细胞主要产生T细胞生长因子,白细胞介素2(IL-2)。抗原性刺激可以导致这些细胞的分化,有时候分化成叫做Th0的群体,其产生细胞因子,并且随后分化成叫做Th1和Th2的亚类,它们具有相对限制的细胞因子产生和效应子功能谱。Th1细胞分泌γ干扰素(IFN-γ)、白细胞介素-2(IL-2),其活化巨噬细胞并且是对抗胞内微生物的细胞介导的免疫以及迟发型超敏反应的主要效应子。受Th1细胞刺激的抗体同种型可以有效地激活补体和调理抗原用于吞噬。因此,Th1细胞触发吞噬细胞介导的宿主防御。胞内微生物感染往往诱导幼稚T细胞分化成Th1亚类,这促进吞噬细胞对微生物的清除。另一方面,Th2细胞产生白细胞介素-4(IL-4)(刺激IgE抗体产生)、白细胞介素-5(IL-5)(为嗜酸性粒细胞-活化因子)、和白细胞介素-10(IL-10)与白细胞介素-13(IL-13)(与白细胞介素-4(IL-4)一起抑制细胞介导的免疫)。因此,Th2细胞主要负责:由IgE和嗜酸性粒细胞介导的不依赖吞噬细胞的宿主防御,例如针对某些蠕虫寄生物;以及由肥大细胞和嗜碱性粒细胞的IgE依赖性激活而导致的变应性反应(ABBAS A.K等人,Cellularand molecular Immunology,W.B.Saunders Co.)。
Winkler等人(WINKLER,S.,M.Willheim,K.Baier,等人.1998.在恶性疟原虫(Plasmodium falciparum)疟疾中在寄生物血症清除期间产生Th1和Th2细胞因子的T细胞的相互调节作用(Reciprocal regulation ofTh1-and Th2-cytokine-producing T cells during clearance of parasitemiain Plasmodium falciparum malaria),Infect.Immun.66:6040-6044.)已经在非并发恶性疟原虫疟疾患者中显示IFN-γ在人抗疟疾宿主防御中作为关键分子的作用,并且他们不支持白细胞介素-4(IL-4)直接参与恶性疟原虫寄生物的清除。另外,已经显示,对于同一种给定抗原,佐剂可以在抗体应答期间导致朝向优势同种型的偏移(TOELLNER K.-M.等人.J.Exp.Med.1998,187:1193)。例如,已知铝盐如Alhydrogel在小鼠中诱导基本上Th2型应答并且促进IgG1形成或甚至IgE形成(ALLISON A.C.在《Vaccine design-The role of cytokine networks》中,第293卷,1-9PlenumPress 1997),这可能在带有变应性素因的受试者中带来问题。
就此方面而言,目前依然需要获得能够使免疫应答朝向针对抗原的Th1型应答的佐剂。
发明公开
本申请人已经惊讶地发现,特定的酿酒酵母线粒体核酸级分是TLR激活物并且能够使免疫应答朝向针对抗原的Th1型应答。
如完整申请书通篇范围内所用,″Th1型应答″指刺激γ干扰素(IFN-γ)、白细胞介素-2(IL-2)和/或白细胞介素-12(IL-12)产生的应答。
如完整申请书通篇范围内所用,除非上下文另有清楚说明,否则″一(a)″和″一个(an)″以这样的意思使用,从而它们意指″至少一个″、″至少第一″、″一个或多个″或″复数个″所指的组分或步骤。
如完整申请书通篇范围内所用,无论在本文中何处使用,“和/或”包括“和”、“或”和“由该术语连接的要素的所有或任何其他组合”的意思。
如完整申请书通篇范围内所用,“包含”和“包括”意指产品、组合物和方法包括所指的组分或步骤,但不排除其他组分或步骤。当用来定义产品、组合物和方法时,“基本上由......组成”应意指排除具有任何实质意义的其他组分或步骤。因此,基本上由所提及组分组成的组合物不排除痕量杂质和可药用载体。“由......组成”应当意指排除除痕量要素外的其他组分或步骤。
本发明涉及酿酒酵母线粒体核酸级分和抗原用于制备意在使免疫应答朝向针对所述抗原的Th1型应答的药物组合物的用途,特征在于所述的酿酒酵母线粒体核酸级分由包括以下步骤的方法制备:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分。
酿酒酵母(S.c.)被充分地描述(Meyen ex E.C.Hansen,1883)并且是市售的(例如S.c.DSM No.1333 ATCC 9763;S.c.DSM No.70464 NCYC1414;S.c.DSM No.2155 ATCC 7754;S.c.DSM No.70869;S.c.DSM No.70461 NCYC 1412;S.c.AH109 Clontech;S.c.Y187 Clontech;S.c.W303Biochem)。在本发明的优选实施方案,所用的酿酒酵母是如实施例1中所述的酿酒酵母AH109(Clontech)。在本发明的另一个优选实施方案,所用的酿酒酵母是如实施例1中所述的酿酒酵母W303(Biochem)。
用于步骤a)中培养酿酒酵母的方法是本领域技术人员熟知的(Guthrie,C.和Fink,G.R.(1991)酵母遗传学与分子生物学指南(Guide toyeast genetics and molecular biology)-Methods in Enzymology(AcademicPress,San Diego,CA)194:1-932 Heslot,H.和Gaillardin,C.,编著.(1992)酵母的分子生物学与基因工程(Molecular Biology and Genetic Engineeringof Yeasts),CRC Press,Inc.)。允许酿酒酵母生长的培养基被充分地描述(例如培养基1017YPG培养基DSMZ;培养基186YM培养基DSMZ;培养基393YPD培养基DSMZ)并且某些是市售的(例如YPD培养基Clontech)。允许酿酒酵母生长的培养基包含至少酵母提取物、胨和葡萄糖。所用的培养基可以补充有一种或多种营养素,例如氨基酸、维生素、盐和/或其它。它们中一些是市售的(例如与补充有腺嘌呤的YPD培养基相对应的YPDA培养基Clontech)。培养条件例如营养素、温度和持续时间是本领域技术人员熟知的(Guthrie,C.和Fink,G.R.(1991)酵母遗传学与分子生物学指南(Guide to yeast genetics and molecular biology)-Methods in Enzymology(Academic Press,San Diego,CA)194:1-932 Heslot,H.和Gaillardin,C.,编著.(1992)酵母的分子生物学与基因工程(Molecular Biology and GeneticEngineering of Yeasts),CRC Press,Inc.)。在本发明的优选实施方案中,使用如实施例1中所述的方法和条件,其中酿酒酵母AH109或W303在补充有腺嘌呤(100μg/ml)的包含酵母提取物(1%)、胨(1%)和葡萄糖(2%)的培养基中在28℃和30℃之间的温度培养。
步骤a)中离心前面所获得的酿酒酵母培养物可以在加速度下以适于沉淀全部酿酒酵母的时间进行。本领域技术人员能够确定哪个速度和哪个持续时间是最合适的。对于步骤a)中离心前面所获的酿酒酵母培养物,优选地如实施例1中所述在3500转/分钟的加速度下进行至少15分钟。
破碎a)步骤中所获得的酿酒酵母沉淀的步骤b)可以通过本领域技术人员熟知的方法、手段和任意系统或装置实施(例如RIEDER SE,Emr SD,酿酒酵母的亚细胞分级分离方法概览(Overview of subcellularfractionation procedures for the yeast Saccharomyces cerevisiae),CurrProtoc Cell Biol.2001年5月;第3章:第3.7单元;RIEDER SE,Emr SD,从酿酒酵母分离亚细胞级分(Isolation of subcellular fractions from theyeast Saccharomyces cerevisiae),Curr Protoc Cell Biol.2001年5月;第3章:第3.8单元;HARJU S,Fedosyuk H,Peterson KR.,快速分离酵母基因组DNA(Rapid isolation of yeast genomic DNA):Bust n′Grab,BMCBiotechnol.2004月1日;4:8.),如使用研钵及研杵的手工破碎;使用涡旋器(例如桌面涡旋混合仪Top Mix 94323 Bioblock Scientifique)在优选具有0.1与5mm之间直径并且更优选0.7mm直径的玻璃珠存在下破碎;使用(例如从Labnet可商业获得的)涡旋混合仪破碎;使用(例如从Kontes可商业获得的)Dounce匀浆器、使用(例如从Kontes可商业获得的)Potter-Elvehjem匀浆器或使用SLM Aminco弗氏细胞压碎器,通过基于液体的匀浆法破碎;使用(例如从Brinkmann Instruments可商业获得的)Waring Blender Polytron进行机械破碎;通过使用超声波仪(例如从Biologics;Misonix;GlenMills可商业获得)的超声破碎法破碎;或通过冻融法破碎。破碎a)步骤中所获的酿酒酵母沉淀的步骤b)优选地在4℃温度进行。明显地根据待处理的a)步骤中获得的酿酒酵母沉淀物的初始量,本领域技术人员能够确定哪一种前述破碎法是最合适的。本领域技术人员还能够确定步骤b)中的破碎条件,例如速度和持续时间。在本发明的优选实施方案中,破碎a)步骤中所获的酿酒酵母沉淀的步骤b)通过在玻璃珠存在下使用涡旋器破碎而进行。玻璃珠优选地具有0.1与5mm之间直径并且更优选地0.7mm直径。优选地按1至20个循环、更优选5个循环,每个循环30秒至2分钟、更优选每个循环1分钟,进行破碎。在本发明的更优选实施方案中,破碎a)步骤中所获酿酒酵母沉淀的步骤b)通过在玻璃珠存在下使用涡旋器破碎而进行,其中玻璃珠具有0.7mm直径,并且其中以5个循环且每个循环1分钟持续时间,如实施例1中所述进行破碎。
破碎a)步骤中所获得酿酒酵母沉淀可以在蛋白酶存在下通过消化作用而进行。根据本发明优选使用的蛋白酶是来自酵母细胞壁的β-葡聚糖酶例如(内切或外切)β-1,3-葡聚糖酶或(内切或外切)β-1,4-葡聚糖酶,包括但不限于酶解酶(zymolyase)和oxalyticase。根据本发明,优选地调节反应条件、溶液pH、反应的温度及持续时间至最适于所选蛋白酶的活性的条件。本领域技术人员能够确定这些条件(RIEDER SE,Emr SD,酵母酿酒的亚细胞分级方法概览(Overview of subcellular fractionation procedures forthe yeast Saccharomyces cerevisiae),Curr Protoc Cell Biol.2001年5月;第3章:第3.7单元;RIEDER SE,Emr SD,从酵母酿酒分离亚细胞级分(Isolation of subcellular fractions from the yeast Saccharomyces cerevisiae),Curr Protoc Cell Biol.2001年5月;第3章:第3.8单元)。在本发明的另一个优选实施方案中,破碎a)步骤中所获得酿酒酵母沉淀的步骤b)因而在一种或多种蛋白酶、优选酶解酶或oxalyticase或其组合的存在下消化a)步骤中所获得的酿酒酵母沉淀而进行。
离心b)步骤中获得的混合物的步骤c)在加速度下以适于沉淀膜碎片以及细胞核的持续时间进行。本领域技术人员能够确定哪个速度和哪个持续时间是最合适的。离心b)步骤中获得的混合物的步骤c)优选地如实施例1中所述在4000转/分钟的加速度下进行10分钟。离心b)步骤中获得的混合物的步骤c)优选地在4℃温度进行。
超速离心c)步骤中所获得上清液的步骤d)在加速度下以适于沉淀线粒体的持续时间进行。本领域技术人员能够确定哪个速度和哪个持续时间是最合适的。超速离心c)步骤中所获得上清液的步骤d)优选地如实施例1中所述在39000转/分钟的加速度下进行90分钟。超速离心c)步骤中所获得上清液的步骤d)优选地在4℃温度进行。
用于提取核酸的方法是本领域技术人员熟知的。从包含d)步骤中所获线粒体的沉淀物中提取核酸的步骤e)可以通过例如酚-二氯甲烷提取法或酚-氯仿提取法(例如CHOMCZYNSKI P.和Sacchi N.(1987),“通过硫氰酸胍-酚-氯仿提取的单步骤RNA分离法(Single Step Method of RNAIsolation by Acid Guanidinium Thiocyanate-Phenol-ChloroformExtraction)”Anal.Biochem.162:156-159)进行。在本发明的优选实施方案中,使用如实施例1中所述的方法和条件,其中从包含d)步骤中所获线粒体的沉淀中提取核酸的步骤e)可以通过酚-二氯甲烷提取而进行。
从e)步骤中获得的上清液回收核酸级分的步骤f)通过本领域技术人员熟知的醇沉淀法(例如HARJU S,Fedosyuk H,Peterson KR.,快速分离酵母基因组DNA(Rapid isolation of yeast genomic DNA):Bust n′Grab,BMC Biotechnol.年4月1日;4:8.)进行。在本发明的优选实施方案中,使用如实施例1中所述的方法和条件,其中从e)步骤中获得的上清液回收核酸级分的步骤f)可以通过醇沉淀以进行。
在步骤f)中回收的核酸级分包含线粒体核糖核酸(RNA)。如实施例2中显示(图1),该酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)是RNA酶敏感的。如实施例3中显示(表3),该酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)的生物学特性在RNA酶存在下消失。
就此而言,包含于本发明酿酒酵母线粒体核酸级分中的核酸优选地是RNA。
本发明的酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)能够与人TLR结合。本领域技术人员能够通过使用本领域中可获得的技术如实施例3中所述的那些技术,确定核酸与TLR结合的能力。在本发明的更优选实施方案中,如实施例3中所述的,本发明的酿酒酵母线粒体核酸级分能够与人TLR3、TLR4和TLR7结合。
本发明的酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)旨在使免疫应答朝向针对抗原的Th1型应答。更具体地,本发明的酿酒酵母线粒体核酸级分意图诱导产生针对抗原的γ干扰素(IFN-γ)、白细胞介素-2(IL-2)和/或白细胞介素-12(IL-12)。本领域技术人员能够通过使用本领域中可获得的技术如实施例4和实施例6中所述的那些技术,确定核酸诱导γ干扰素(IFN-γ)、白细胞介素-2(IL-2)和白细胞介素-12(IL-12)产生的能力。在本发明的更优选实施方案中,本发明的酿酒酵母线粒体核酸级分意在诱导产生
-如实施例4和实施例6中所述并且分别在图2和图4中显示的,γ干扰素(IFN-γ);
-如实施例6中所述并且在图5中显示的,白细胞介素-12(IL-12)。
如实施例6中所述并且在图6中显示,本发明的酿酒酵母线粒体核酸级分不能够诱导α干扰素(IFN-α)产生。
如完整申请书通篇范围内所用,″抗原″指含有一个或多个能刺激宿主免疫系统以产生抗原特异性体液和/或细胞应答的表位的分子。该术语与术语“免疫原”可互换地使用。抗体,如抗独特型抗体,或其片段和可以模拟抗原或抗原决定簇的合成性肽模拟表位也落入本文中所用的抗原定义中。
根据本发明,抗原优选地选自由肿瘤相关抗原、传染性生物特异性抗原和变应原特异性抗原组成的组。
根据本发明的第一实施方案,抗原是肿瘤相关抗原。如完整申请书通篇范围内所用,“肿瘤相关抗原”(TAA)指在肿瘤细胞中比在相同组织类型的非肿瘤细胞中以更高的频率或密度被检测到的分子。TAA的实例包括但不限于CEA、MART-1、MAGE-1、MAGE-3、GP-100、MUC-1(见例如WO92/07000;EP554344;US5,861,381;US6,054,438;WO98/04727;WO98/37095)、MUC-2、点突变的ras癌基因、正常或点突变的p53、过量表达的p53、CA-125、PSA、C-erb/B2、BRCA I、BRCA II、PSMA、酪氨酸酶、TRP-1、TRP-2、NY-ESO-1、TAG72、KSA、HER-2/neu、bcr-abl、pax3-fkhr、ews-fli-1、存活蛋白(survivin)、合胞素(syncytin)(例如合胞素-1,见例如WO99/02696;WO2007/090967;US6,312,921)、间皮素(mesothelin)和LRP。这些分子的序列已经在本领域中描述。在本发明的优选实施方案中,抗原是TAA MUC-1。实施例5描述了本发明的酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原用于制备治疗癌症的药物组合物的用途。
根据本发明的另一个实施方案,抗原是传染性生物特异性抗原。如完整申请书通篇范围内所用,“传染性生物特异性抗原”指病毒、细菌、真菌或寄生物的特异性抗原。
如完整申请书通篇范围内所用,“病毒”包括,但不限于,逆转录病毒科、小RNA病毒科(例如脊髓灰质炎病毒、甲型肝炎病毒;肠病毒,人柯萨奇病毒、鼻病毒、埃可病毒);杯状病毒科(例如引起胃肠炎的病毒株);披膜病毒科(例如马脑炎病毒、风疹病毒);黄病毒科(例如登革病毒、脑炎病毒、黄热病毒);冠状病毒科(例如冠状病毒);弹状病毒科(例如水泡性口炎病毒、狂犬病病毒);线状病毒科(例如埃博拉病毒);副粘病毒科(例如副流感病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞体病毒);正粘病毒科(例如流感病毒);布尼亚病毒科(例如汉坦病毒、bunga病毒、白蛉病毒和纳伊罗病毒);沙粒病毒科(出血热病毒);呼肠孤病毒科(例如呼肠孤病毒、环状病毒和轮状病毒);双RNA病毒科;嗜肝DNA病毒科(乙型肝炎病毒);细小病毒科(细小病毒);乳多空病毒科(乳头瘤病毒、多瘤病毒);腺病毒科(大部分腺病毒);疱疹病毒科(单纯疱疹病毒(HSV)1和2、水痘-带状疱疹病毒、巨细胞病毒(CMV)、疱疹病毒;痘病毒科(天花病毒、痘苗病毒、痘病毒);和虹彩病毒科(例如非洲猪瘟病毒)。病毒抗原包括例如来自肝炎病毒A、B、C、D和E、HIV、疱疹病毒、巨细胞病毒、水痘带状疱疹病毒、乳头瘤病毒、E-B病毒、流感病毒、副流感病毒、腺病毒、柯萨奇病毒、小RNA病毒、轮状病毒、呼吸道合胞体病毒、痘病毒、鼻病毒、风疹病毒、乳多空病毒、细小病毒、腮腺炎病毒、麻疹病毒的抗原。已知病毒抗原的一些非限制性实例包括:HIV-1特异性抗原如tat、nef、gp120或gp160、gp40、p24、gag、env、vif、vpr、vpu、rev或其部分和/或组合;人疱疹病毒特异性抗原如gH、gL、gM、gB、gC、gK、gE或gD或其部分和/或组合,或来自HSV1或HSV2的立即早期蛋白如ICP27、ICP47、ICP4、ICP36;巨细胞病毒、尤其人巨细胞病毒特异性抗原,如gB或其衍生物;E-B病毒特异性抗原如gp350或其衍生物;水痘带状疱疹病毒特异性抗原如gpl、11、111和IE63;肝炎病毒特异性抗原如乙型肝炎、丙型肝炎或戊型肝炎病毒抗原(例如HCV的env蛋白E1或E2、核心蛋白、NS2、NS3、NS4a、NS4b、NS5a、NS5b、p7或其部分和/或组合);人乳头瘤病毒特异性抗原(例如HPV6、11、16、18、例如L1、L2、E1、E2、E3、E4、E5、E6、E7或其部分和/或组合);其他病毒病原体如呼吸道合胞体病毒(例如F和G蛋白或其衍生物)、副流感病毒、麻疹病毒、腮腺炎病毒、黄病毒(例如黄热病病毒、登革病毒、蜱传脑炎病毒、日本脑炎病毒)或流感病毒感染细胞(例如HA、NP、NA或M蛋白或其部分和/或组合)特异性抗原。本发明特别包括与p53的结合被改变或至少被显著降低的任何HPV E6多肽的使用、和/或与Rb的结合被改变或至少被显著降低的任何HPV E7多肽的使用(MUNGER等人.人乳头瘤病毒E7蛋白与成视网膜细胞瘤肿瘤抑制基因产物的复合物形成(Complex formation of human papillomavirus E7proteins with the retinoblastoma tumor suppressor gene product).TheEMBO journal.1989,第8卷,第13期,第4099-4105页;CROOK,等人.p53的降解可以通过与p53结合和反式激活所需要的那些HPV E6序列不同的HPV E6序列靶向.Cell.1991,第67卷,第3期,第547-556页;HECK等人.结合成视网膜细胞瘤蛋白的效率与人乳头瘤病毒E7癌蛋白质的转化能力相关(E fficiency of binding the retinoblastoma protein correlateswith the transforming capacity of the E7oncoproteins of the humanpapillomaviruses).Proc.Natl.Acad.Sci.U.S.A..1992,第89卷,第10期,第4442-4446页;PHELPS,等人.人乳头瘤病毒16型E7癌蛋白质的结构-功能分析(Structure-function analysis of the human papillomavirus type16E7oncoprotein).Journal of Virology.1992,第66卷,第4期,第2418-2427页)。适用于本发明目的非致癌性HPV-16E6变体缺失位于大约第118位置至大约第122位置(+1代表天然HPV-16E6多肽的第一个甲硫氨酸残基)之间的一个或多个氨基酸残基,特别优选完全缺失第118至第122位残基(CPEEK)。适用于本发明目的非致癌性HPV-16E7变体缺失位于大约第21位置至大约第26位置(+1代表天然HPV-16E7多肽的第一个氨基酸残基)之间的一个或多个氨基酸残基,特别优选完全缺失第21至第26位残基(DLYCYE)。根据优选的实施方案,进一步修饰用于本发明中的一种或多种HPV-16早期多肽,从而改善I类MHC和/或II类MHC呈递作用和/或刺激抗HPV免疫。HPV E6和E7多肽是核蛋白并且先前已经被证实膜呈递可以改善它们的治疗效力(见例如WO 99/03885)。因而,可以有利的是,修饰至少一种HPV早期多肽,从而将其锚定至细胞膜上。可以通过在HPV早期多肽中并入膜锚定序列以及,如果该天然多肽缺少分泌序列的话,分泌序列(即,信号肽),容易地实现膜锚定作用。膜锚定序列和分泌序列是本领域已知的。简而言之,分泌序列存在于膜呈递或分泌型多肽的氨基端并且启动它们进入内质网(ER)。它们通常包含15至35个基本上疏水的氨基酸,所述氨基酸随后由位于ER的特定内肽酶去除以产生成熟的多肽。膜锚定序列通常是本质上高度疏水的并且起到在细胞膜中锚定多肽的作用(见例如Branden等人,1991,蛋白质结构导论(Introduction to Protein Structure),NY GARLAND,1991.第202-214页)。可以在本发明中使用的膜锚定序列和分泌序列是多种多样的。它们可以从包含该序列的任何膜锚定或分泌性多肽(例如细胞多肽或病毒多肽)如狂犬病病毒糖蛋白、HIV病毒包膜糖蛋白或麻疹病毒F蛋白获得,或者可以是合成的。在根据本发明使用的各种早期HPV-16多肽中插入的膜锚定序列和/或分泌序列可以具有共同或不同来源。分泌序列的优选插入位点是翻译起始密码子的氨基端下游,膜锚定序列的优选插入位点是羧基端,例如终止密码子的紧邻上游。用于本发明中的HPV E6多肽优选地通过插入麻疹病毒F蛋白的分泌信号和膜锚定信号进行修饰。用于本发明中的HPV E7多肽优选地通过插入狂犬病病毒糖蛋白的分泌信号和膜锚定信号进行修饰。就此而言,在本发明的优选实施方案中,抗原是人乳头瘤病毒(HPV)特异性抗原,优选地是HPV-16或/和HPV-18特异性抗原,并且更优选地是选自由HPV-16或/和HPV-18的E6早期编码区、HPV-16或/和HPV-18的E7早期编码区和其部分或组合组成的组中的抗原。实施例4描述了本发明酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)和HPV16E7抗原用于制备使免疫应答朝向针对HPV16E7抗原的Th1型应答的药物组合物的用途。
如完整申请书通篇范围内所用,“细菌”包括革兰氏阳性和革兰氏阴性细菌。革兰氏阳性细菌包括,但不限于巴氏杆菌属(Pasteurella)物种、葡萄球菌属(Staphylococci)物种和链球菌属(Streptococcus)物种。革兰氏阴性细菌包括,但不限于大肠杆菌(Escherichia coli)、假单胞菌属(Pseudomonas)物种和沙门氏菌属(Salmonella)物种。感染性细菌的具体实例包括但不限于幽门螺杆菌(Helicobacter pyloris)、伯氏疏螺旋体(Borreliaburgdorferi)、嗜肺军团菌(Legionella pneumophilia)、分枝杆菌属物种(Mycobacteria sps)(例如结核分枝杆菌、鸟分枝杆菌(M.avium)、胞内分枝杆菌(M.intracellulare)、堪萨斯分枝杆菌(M.kansaii)、戈登分枝杆菌(M.gordonae))、金黄色葡萄球菌(Staphylococcus aureus)、淋病奈瑟菌(Neisseria gonorrhoeae)、脑膜炎奈瑟菌(Neisseria meningitidis)、单核细胞增生性李斯特氏菌(Listeria monocytogenes)、化脓链球菌(Streptococcuspyogenes)(A组链球菌)、无乳链球菌(Streptococcus agalactiae)(B组链球菌)、链球菌(草绿色组)、粪链球菌(Streptococcus faecalis)、牛链球菌(Streptococcus bovis)、链球菌(厌氧物种)、肺炎链球菌、致病性弯曲杆菌属(Campylobacter)物种、肠球菌属(Enterococcus)物种、流感嗜血杆菌(Haemophilus influenzae)、炭疽芽孢杆菌(Bacillus antracis)、白喉棒状杆菌(Corynebacterium diphtheriae)、棒状杆菌属(Corynebacterium)物种、红斑丹毒丝菌(Erysipelothrix rhusiopathiae)、产气荚膜梭菌(Clostridiumperfringens)、破伤风梭菌(Clostridium tetani)、产气肠杆菌(Enterobacteraerogenes)、肺炎克雷伯菌(Klebsiella pneumoniae)、多杀巴氏菌(Pasturellamultocida)、拟杆菌属(Bacteroides)物种、具核梭杆菌(Fusobacteriumnucleatum)、念珠状链杆菌(Streptobacillus moniliformis)、苍白密螺旋体(Treponema pallidium)、细弱密螺旋体(Treponema pertenue)、钩端螺旋体(Leptospira)、立克次氏体(Rickettsia)和以色列放线菌(Actinomycesisraelli)。
如完整申请书通篇范围内所用,“真菌”包括,但不限于新型隐球菌(Cryptococcus neoformans)、荚膜组织胞浆菌(Histoplasma capsulatum)、粗球孢子菌(Coccidioides immitis)、皮炎芽生菌(Blastomyces dermatitidis)、沙眼衣原体(Chlamydia trachomatis)和白假丝酵母(Candida albicans)。
如完整申请书通篇范围内所用,“寄生物”包括但不限于以下属:疟原虫属(Plasmodium)(例如恶性疟原虫(Plasmodium falciparum)、三日疟原虫(Plasmodium malariae)、疟原虫属物种(Plasmodium spp.)、卵形疟原虫(Plasmodium ovale)或间日疟原虫(Plasmodium vivax))、巴贝虫属(Babesia)(例如微小巴贝虫(Babesia microti)、巴贝虫属物种(Babesia spp)或分歧巴贝虫(Babesia divergens))、利什曼原虫属(Leishmania)(例如热带利什曼原虫(Leishmania tropica)、利什曼原虫某些种(Leishmania spp.)、巴西利什曼原虫(Leishmania braziliensis)或杜氏利什曼原虫(Leishmaniadonovani)、锥虫属(Trypanosoma)(例如冈比亚锥虫(Trypanosomagambiense)、锥虫属某些种(Trypanosoma spp.)、引起非洲睡眠病的罗德西亚锥虫(Trypanosoma rhodesiense)或引起查格斯病的克氏锥虫(Trypanosoma cruzi)和弓形体属(Toxoplasma)(例如鼠弓形体(Toxoplasmagondii))。
如完整申请书通篇范围内所用,“变应原”指可以在易感受试者中诱导变态或哮喘反应的物质。变应原包括,但不限于花粉、昆虫毒液、动物毛皮尘、真菌孢子和药物(例如青霉素)。天然的动物和植物变应原的实例包括,但不限于以下属的特异性蛋白质:犬属(Canine)(家犬(Canisfamiliaris));尘螨属(Dermatophagoides)(例如,尘螨(Dermatophagoidesfarinae));猫属(Felis)(例如,家猫(Felis domesticus));豚草属(Ambrosia)(例如,豚草(Ambrosia artemiisfolia));黑麦草属(Lolium)(例如,宿根黑麦草(Lolium perenne)或多花黑麦草(Lolium multiflorum);柳杉属(Cryptomeria)(例如,日本柳杉(Cryptomeria japonica));链格孢属(Alternaria)(例如,链格孢(Alternaria alternata));赤杨属(Alder);桤木属(Alnus)(例如,欧洲桤木(Alnus gultinoasa));桦木属(Betula)(例如,垂枝桦(Betula verrucosa));栎属(Quercus)(例如,白栎(Quercus alba));木犀榄属(Olea)(例如,油橄榄(Olea europa));蒿属(Artemisia)(例如,艾蒿(Artemisia vulgaris));车前草属(Plantago)(例如,长叶车前草(Plantagolanceolata));墙草属(Parietaria)(例如,墙草(Parietaria officinalis)或欧蓍草(Parietaria judaica));小蠊属(Blattella)(例如,德国小蠊(Blattellagermanica);蜜蜂属(Apis)(例如,Apis multiflorum);柏木属(Cupressus)(例如,地中海柏木(Cupressus sempervirens)、绿干柏(Cupressus arizonica)和大果柏木(Cupressus macrocarpa));刺柏属(Juniperus)(例如,Juniperussabinoides、铅笔柏(Juniperus virginiana)、普通柏(Juniperus communis)和阿希刺柏(Juniperus ashei));侧柏属(Thuya)(例如,侧柏(Thuyaorientalis));扁柏属(Chamaecyparis)(例如,日本扁柏(Chamaecyparisobtusa));大蠊属(Periplaneta)(例如,美洲大蠊(Periplaneta americana));冰草属(Agropyron)(例如,匍匐冰草(Agropyron repens));黑麦属(Secale)(例如,黑麦(Secale cereale));小麦属(Triticum)(例如,普通小麦(Triticum aestivum));鸭茅属(Dactylis)(例如,鸭茅(Dactylis glomerata));羊茅属(Festuca)(例如,牛尾草(Festuca elatior));早熟禾属(Poa)(例如,草地早熟禾(Poa pratensis)或加拿大早熟禾(Poa compressa));燕麦属(Avena)(例如,燕麦(Avena sativa));绒毛草属(Holcus)(例如,绒毛草(Holcuslanatus));黄花茅属Anthoxanthum(例如,黄花茅(Anthoxanthumodoratum));燕麦草属(Arrhenatherum)(例如,燕麦草(Arrhenatherumelatius));剪股颖属(Agrostis)(例如,小糠草(Agrostis alba));梯牧草属(Phleum)(例如,梯牧草(Phleum pratense));虉草属(Phalaris)(例如,虉草(Phalaris arundinacea));雀稗草属(Paspalum)(例如,百喜草(Paspalumnotatum));蜀黍属(Sorghum)(例如,石茅高粱(Sorghum halepensis));和雀麦属(Bromus )(例如,无芒雀麦(Bromus inermis))。
根据本发明,抗原优选地选自由肽、核酸(例如DNA或RNA或其杂合体)、脂质、脂肽和糖(例如寡糖或多糖)组成的组。抗原也可以是能够特异地引导免疫应答指向针对选自下述的抗原的Th1型应答的任何化合物:肿瘤相关抗原、传染性生物特异性抗原或变应原特异性。
根据本发明的优选实施方案,抗原包含于载体中。根据本发明,该载体优选地选自质粒或病毒载体。
就质粒而言,可以提到例如从pBR322(Gibco BRL)、pUC(GibcoBRL)、pBluescript(Stratagene)、pREP4、pCEP4(Invitrogene)或pPoly(LATHE,等人.用于切除完整插入物的质粒和噬菌体载体(Plasmidand bacteriophage vectors for excision of intact inserts).Gene.1987,第57卷,第2-3期,第193-201页)获得的质粒。通常而言,质粒是技术人员已知的,并且尽管它们许多是市售的(例如,前面提到的质粒),但也可以使用遗传操作技术修饰它们或构建它们。优选地,可以在本发明中使用的质粒含有确保在生产细胞和/或宿主细胞中起始复制的复制起点(例如,可以选择ColE1起点用于旨在于大肠杆菌中生产的质粒,且如果希望质粒在哺乳动物宿主细胞中自我复制,则可以选择oriP/EBNA1系统,LUPTON,等人.标定促进Epstein-Barr病毒衍生性质粒在人细胞中染色体外维持的Epstein-Barr病毒遗传元件(Mapping genetic elements of Epstein-Barrvirus that facilitate extrachromosomal persistence of Epstein-Barrvirus-derived Plasmids in human cells).Molecular and cellular biology.1985,第5卷,第10期,第2533-42页.;YATES,等人.从Epstein-Barr病毒衍生的质粒在多种哺乳动物细胞中的稳定复制.Nature.1985,第313卷,第6005期,第812-815页)。质粒可以额外地包含能够选择或鉴定转染细胞的选择基因(弥补营养缺陷型突变、编码抗生素耐药性的基因等)。自然地,质粒可以含有改善其在给定细胞中的维持和/或其稳定性的额外元件(促进质粒以单体形式维持的cer序列(SUMMERS等人.高拷贝数质粒的多聚化造成不稳定:ColE1编码对质粒单体化和和稳定性必需的决定因子(Multimerization of high copy number plasmids causes instability:ColE 1encodes a determinant essential for plasmid monomerization and stability).Cell.1984,第36卷,第4期,第1097-1103页),用于整合至细胞基因组内的序列)。
就病毒载体而言,可以提及例如从痘病毒、从腺病毒、从逆转录病毒、从疱疹病毒、从甲病毒、从泡沫病毒或从腺相关病毒获得的病毒载体。可以使用有复制能力的或复制缺陷的病毒载体。“有复制能力的病毒载体”指在任何反式互补作用不存在的情况下能够在宿主细胞中复制的病毒载体。“复制缺陷型病毒载体”指在没有某种形式的反式互补的情况下不能够在宿主细胞中复制的病毒载体。另外,优选使用不整合的载体。就这个方面而言,腺病毒载体和从痘病毒获得的载体特别适于实施本发明。
本发明的优选实施方案中,病毒载体从痘病毒、优选地从痘苗病毒并且更优选地从改良安卡拉痘苗病毒(MVA)或其衍生物获得。“衍生物”指这样的病毒,其显示与保藏株具有基本上相同的复制特征,但是在其基因组的一个或多个部分中显示差异。
如完整申请书通篇范围内所用,“痘苗病毒”(VV)包括但不限于VV株Dairen I、IHD-J、L-IPV、LC16M8、LC16MO、Lister、LIVP、Tashkent、WR 65-16、Wyeth、Ankara、Copenhagen、Tian Tan、Western Reserve(WR)及其衍生物,例如包含缺陷型F2L基因的VV(见WO2009/065547)和包含缺陷型I4L和/或F4L基因的VV(见WO2009/065546)。VV含有一个大的双链体DNA基因组(187千碱基对),是在感染细胞的细胞质中复制的唯一已知DNA病毒家族的成员。VV在欧洲专利EP83286中充分描述。VV哥本哈根株的基因组已经作图并测序(Goebel等人.,1990,Virol.179,247-266和517-563;Johnson等人.,1993,Virol.196,381-401)。
如完整申请书通篇范围内所用,“改良安卡拉痘苗病毒(MVA)”指通过VV安卡拉株(CVA)在CEF上连续传516代产生的高度减毒的VV病毒(Mayr,A.,等人。Infection 3,6-14,1975)及其衍生物。MVA病毒以保藏号N602I-721在法国国家培养物与微生物保藏中心(Collection Nationalede Cultures de Microorganismes(CNCM))保藏。MVA载体和产生此类载体的方法在欧洲专利EP 83286A和EP 206920A、在国际申请WO07/147528中以及在SUTTER,等人.非复制型疫苗载体高效表达重组基因(Nonreplicating vaccinia vector efficiently expresses recombinant genes).Proc.Natl.Acad.Sei.U.S.A..1992,第89卷,第22期,第10847-10851页中充分描述。MVA的基因组已经作图并测序(Antoine等人.,1998,Virol.244,365-396)。根据更优选的实施方案,抗原可以在MVA载体的缺失区I、II、III、IV、V和VI中并且甚至更优选在缺失区III中插入(MEYER等人.标定高度减毒的痘苗病毒MVA的基因组中的缺失区和它们对毒力的影响(Mapping of deletions in the genome of the highly attenuated vacciniavirus MVA and their influence on virulence).The Journal of generalvirology.1991,第72卷,第Pt5期,第1031-1038页;SUTTER等人.从宿主范围受限和高度减毒的痘苗病毒MVA株中衍生的重组载体在小鼠中刺激针对流感病毒的保护性免疫(A recombinant vector derived from thehost range-restricted and highly attenuated MVA strain of vaccinia virusstimulates protective immunity in mice to influenza virus).Vaccine.1994,第12卷,第11期,第1032-1040页)。实施例5描述了本发明的酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原用于制备治疗癌症的药物组合物的用途,其中MUC-1抗原包含于MVA载体中。
在本发明的另一个优选实施方案中,病毒载体从腺病毒、腺病毒相关病毒、逆转录病毒、疱疹病毒、甲病毒或泡沫病毒或其衍生物获得。
根据本发明使用的腺病毒载体优选地是这样的腺病毒载体,其缺少对复制必需的、选自E1、E2、E4和L1-L5区的至少一个区域的全部或部分以避免该载体在宿主生物或环境中增殖。优选缺失E1区。然而,它可以与(一或多种)其他修饰-/缺失组合,其中所述的其他修饰-/缺失影响尤其是E2、E4和/或L1-L5区的全部或部分,从而使得借助互补性细胞系和/或辅助病毒可以反式地弥补这些缺陷型必需功能。在这个方面,可以使用本领域的第二代载体(见,例如国际申请号94/28152和WO 97/04119)。举例来说,缺失E4转录单位和E1区的主要部分是特别有利的。出于增加克隆容量的目的,腺病毒载体可以额外地缺失非必需E3区的全部或部分。根据另一个备选方案,可以利用最小腺病毒载体,所述的最小腺病毒载体保留对衣壳化所必需的序列,即5′和3′ITR(倒置末端重复序列)和衣壳化区域。多种腺病毒载体和用于制备这些载体的技术是已知的(见,例如GRAHAM,等人Methods in molecular biology.MURREY编著.Thehuman press inc,第109-128页)。从物种来看和从血清型来看,本发明腺病毒载体的来源可以是多种多样的。该载体可以从人或动物(犬、禽、牛、小鼠、绵羊、猪、猴等)来源的腺病毒的基因组获得,或从包含至少两种不同来源的腺病毒基因组片段的杂合体获得。可以更尤其提及的是,犬源CAV-I或CAV-2腺病毒、禽源DAV腺病毒或牛源Bad 3型腺病毒(ZAKHARCHUK等人.减蛋综合征(EDS-76)腺病毒DNA的物理作图和同源性研究(Physical mapping and homology studies of egg drop syndrome(EDS-76)adenovirus DNA).Archives of virology.1993,第128卷,第1-2期,第171-6页;SPIBEY等人.两个犬腺病毒2型毒株的分子克隆和限制性核酸内切酶作图(Molecular cloning and restriction endonucleasemapping of two strains of canine adenovirus type 2).The Journal ofgeneral virology.1989,第70卷,第Pt 1期,第165-72页;JOUVENNE等人.犬腺病毒1和2型基因组的克隆、物理作图和交叉杂交(Cloning,physical mapping and cross-hybridization of the canine adenovirus types 1and 2genomes).Gene.1987,第60卷,第1期,第21-8页;MITTAL等人.开发基于牛腺病毒3型的表达载体(Development of a bovineadenovirus type 3-based expression vector).The Journal of generalvirology.1995,第76卷,第Pt 1期,第93-102页)。然而,优选人源腺病毒载体,该载体优选从C血清型腺病毒、尤其2或5型C血清型腺病毒获得。根据本发明也可以使用有复制能力的腺病毒载体。这些有复制能力的腺病毒载体是本领域技术人员熟知的。在这些载体当中,特别优选在编码55kD P53抑制物的E1b区中缺失的腺病毒载体,如在ONYX-015病毒中(BISCHOFF等人.在p53缺陷型人肿瘤细胞中选择性复制的腺病毒突变体(An adenovirus mutant that replicates selectively in p53-deficienthuman tumor cells).Science.1996,第274卷,第5286期,第373-376页;HEISE等人.展示强力和选择性全身抗肿瘤效力的腺病毒E1A突变体(Anadenovirus E1A mutant that demonstrates potent and selective systemicanti-tumoral efficacy).Nature Medicine.2000,第6卷,第10期,第1134-1139页;WO94/18992)。因此,这种病毒可以用来选择性感染和杀死p53缺陷型赘生细胞。本领域普通技术人员也可以根据已建立的技术,突变并破坏腺病毒5或其他病毒中的p53抑制物基因。也可以在本发明中使用E1A Rb结合区内缺失的腺病毒载体。例如,Δ24病毒是E1A区中携带24个碱基对缺失的突变体腺病毒(FUEYO,等人.靶向Rb途径的突变溶瘤腺病毒在体内产生抗胶质瘤作用(A mutant oncolytic adenovirus targetingthe Rb pathway produces anti-glioma effect in vivo).Oncogene.2000,第19卷,第1期,第2-12页)。Δ24在Rb结合区中具有缺失并且不与Rb结合。因此,该突变体病毒的复制在正常细胞中被Rb抑制。然而,如果Rb失活并且细胞变成致瘤性,则Δ24不再被抑制。取而代之,突变体病毒高效地复制并且裂解Rb缺陷型性细胞。本发明的腺病毒载体可以通过连接或同源性重组(见,例如国际申请号96/17070)或通过在互补性细胞系中重组,而在大肠杆菌(E.coli)中体外产生。
逆转录病毒具有感染并且在大多数情况下整合到正在分裂的细胞中的属性,并且在这个方面是特别适合对癌症使用的。本发明的重组逆转录病毒通常含有LTR序列、衣壳化区、和处于逆转录病毒LTR或内部启动子(如下文所述那些启动子)控制下的本发明核苷酸序列。重组逆转录病毒可以从任何来源(小鼠、灵长类、猫、人等)的逆转录病毒并且尤其从MoMuLV(Moloney小鼠白血病病毒)、MVS(小鼠肉瘤病毒)或Friend小鼠逆转录病毒(Fb29)获得。它在能够反式地供应在病毒粒子构成时所必需的病毒多肽gag、pol和/或env的衣壳化细胞系中增殖。此类细胞系在文献中描述(PA317,Psi CRIP GP+Am-12等)。本发明的逆转录病毒载体可以含有修饰,尤其在LTR(将启动子区替换为真核启动子)或衣壳化区域(替换为异源衣壳化区,例如VL3O型)中,如US 5747323中所述。
根据本发明,载体还包含在抗原是核酸时对于抗原表达所必需的元件。对表达必需的元件可以为能够使核酸序列转录成RNA并且使mRNA翻译成多肽的全部元件。这些元件尤其包括可以是调节型或组成型的启动子。自然,启动子对所选择的载体和宿主细胞是合适的。可以提到的实例是PGK(磷酸甘油酸激酶)基因、MT基因(金属硫蛋白;MCIVOR.人嘌呤核苷磷酸化酶和腺苷脱氨酶:通过使用重组双嗜性逆转录病毒转移基因至培养细胞和小鼠造血干细胞中(Human purine nucleoside phosphorylaseand adenosine deaminase:gene transfer into cultured cells and murinehematopoietic stem cells by using recombinant amphotropic retroviruses).Molecular and cellular biology.1987,第7卷,第2期,第838-46页)、α-1抗胰蛋白酶基因、CFTR基因、表面活性蛋白基因、免疫球蛋白基因、肌动蛋白基因(TABIN等人.将逆转录病毒修改为传递单纯疱疹病毒胸苷激酶基因的真核载体(Adaptation of a retrovirus as a eucaryotic vectortransmitting the herpes simplex virus thymidine kinase gene).Molecularand cellular biology.1982,第2卷,第4期,第426-36页)和SR α基因(TAKEBE等人.SR α启动子:一种由猴病毒40早期启动子和人T细胞白血病病毒1型长末端重复序列的R-U5节段组成的高效和通用型哺乳动物cDNA表达系统(SR alpha promoter:an efficient and versatile mammaliancDNA expression system composed of the simian virus 40early promoterand the R-U5segment of human T-cell leukemia virus type 1long terminalrepeat).Molecular and cellular biology.1988,第8卷,第1期,第466-472页)的真核启动子、SV40病毒(猴病毒)的早期启动子、RSV(劳氏肉瘤病毒)的LTR、HSV-l TK启动子、CMV病毒(巨细胞病毒)早期启动子、痘苗病毒p7.5K、pH5R、pK1L、p28和p11启动子、和E1A和MLP腺病毒启动子。启动子也可以是在肿瘤或癌细胞中刺激表达的启动子。可以具体提到以下基因的启动子:在乳腺癌和前列腺癌中过量表达的MUC-I基因(CHEN,等人.由含有DF3/MUC1启动子的重组腺病毒介导的乳腺癌选择性基因表达和疗法(Breast cancer selective gene expression and therapymediated by recombinant adenoviruses containing the DF3/MUC 1promoter).The Journal of clinical investigation.1995,第96卷,第6期,第2775-82页)、在结肠癌中过量表达的CEA(癌胚抗原)基因(SCHREWE,等人.克隆癌胚抗原的完整基因:其启动子分析显示一个赋予细胞类型特异性表达的区域(Cloning of the complete gene for carcinoembryonicantigen:analysis of its promoter indicates a region conveying celltype-specific expression).Molecular and cellular biology.1990,第10卷,第6期,第2738-48页)、在黑素瘤中过量表达的酪氨酸酶基因(VILE,等人.通过直接肿瘤内注射DNA而利用单纯疱疹病毒胸苷激酶基因的组织特异性表达抑制建立的小鼠黑素瘤的生长(Use of tissue-specific expressionof the herpes simplex virus thymidine kinase gene to inhibit growth ofestablished murine melanomas following direct intratumoral inj ection ofDNA).Cancer res.1993,第53卷,第17期,第3860-4页)、在乳腺癌和胰腺癌中过量表达的ERBB-2基因(HARRIS,等人.使用肿瘤特异性前药激活作用的癌症基因疗法(Gene therapy for cancer using tumour-specificprodrug activation).Gene therapy.1994,第1卷,第3期,第170-5页)、和/或在肝癌中过量表达的甲胎蛋白基因(KANAI,等人.通过腺病毒介导的胞苷脱氨酶基因转移而体内基因治疗产生甲胎蛋白的肝细胞癌(In vivogene therapy for alpha-fetoprotein-producing hepatocellular carcinoma byadenovirus-mediated transfer of cytosine deaminase gene).Cancer res,1997,第57卷,第3期,第461-5页)。巨细胞病毒(CMV)早期启动子是极特别优选的。然而,当使用从痘苗病毒衍生的载体(例如MVA载体)时,特别优选胸苷激酶7.5K基因的启动子。必需元件可以进一步包括改善本发明核苷酸序列在宿主细胞中的表达或其维持的额外元件。具体可以提到内含子序列、分泌信号序列、核定位序列、用于再启动IRES型易位的内部位点、转录终止polyA序列、三联前导序列和复制起点。这些元件是技术人员已知的。
本发明的药物组合物(并且更尤其地是佐剂组合物和疫苗组合物)还可以包含一种或多种改善酿酒酵母线粒体核酸级分和/或抗原的转染效率和/或稳定性的物质。述物质优选地选自由脂质、脂质体、亚微水包油乳液、微粒子、ISCOM和聚合物组成的组。组合物的多种组分可以在宽比率范围内存在。例如,本发明的酿酒酵母线粒体核酸级分和改善酿酒酵母线粒体核酸级分和/或抗原的转染效率和/或稳定性的物质可以按约1∶200至200∶1、优选1∶100至100∶1、更优选约1∶50至50∶1、甚至更优选约1∶10至10∶1、甚至更优选约1∶3至3∶1并且最优选约1∶1比率(体积/体积(v/v)和/或重量/重量(w/w))使用。
如完整申请书通篇范围内所用,“脂质”包括中性、两性、阴离子和/或阳离子脂质。脂质包括,但不限于磷脂(例如天然或合成性磷脂酰胆碱、磷脂酰乙醇胺或磷脂酰丝氨酸)、甘油酯(例如二酰甘油或三酰甘油)、胆固醇、神经酰胺类或脑苷脂类。优选的脂质是阳离子脂质。多种阳离子脂质是本领域已知的,并且其中一些是市售的(例如BALASUBRAMANIAM等人.(1996)Gene Ther.,3:163-172;GAO和HUANG(1995)Gene Ther.,2:7110-7122;US 4,897,355专利;EP 901463B专利和更优选pcTG90)。在本发明的优选实施方案中,脂质是阳离子脂质并且更优选地是如EP 901463B专利中所述的阳离子脂质并且甚至更优选地如EP 901463B专利中所述pcTG90。本发明的酿酒酵母线粒体核酸级分和脂质可以按约1∶200至200∶1、优选1∶100至100∶1、更优选约1∶50至50∶1、甚至更优选约1∶10至10∶1、甚至更优选约1∶3至3∶1并且最优选约1∶1比率(体积/体积(v/v)和/或重量/重量(w/w))使用。
如完整申请书通篇范围内所用,″脂质体″指由双层包围的囊泡,其中所述的双层由如下组分形成,所述组分通常包括脂质,任选地与非脂质组分(例如硬脂胺(Stearylamine))组合。用来形成脂质体的脂质体形成组分可以包括中性、两性、阴离子和/或阳离子脂质。优选的脂质体是阳离子脂质体。阳离子脂质体广泛记录在技术人员可获得的文献中并且其中一些是市售的(例如,FELGNER,等人.阳离子脂质体介导的转染(Cationicliposome mediated transfection).Proceedings of the WesternPharmacology Society.1989,第32期,第115-2页;HODGSON,等人.病毒体阳离子脂质体增强逆转录病毒转导(Virosomes:cationic liposomesenhance retroviral transduction).Nature biotechnology.1996,第14卷,第3期,第339-42页;REMY,等人.采用一系列亲脂性DNA结合分子的基因转移(Gene transfer with a series of lipophilic DNA-binding molecules).Bioconjugate chemistry.1994,第5卷,第6期,第647-54页)。阳离子脂质体(如完整申请书通篇范围内所用)包括,但不限于二油酰磷脂酰乙醇胺(DOPE)、N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)、1,2-双(油酰氧)-3-(三甲基铵基)丙烷(DOTAP)、1,2-双(十六烷基氧)-3-三甲基氨基丙烷(BisHOP)、3[β][N-(N′N′-二甲基氨基乙烷)-氨甲酰基]胆固醇(DC-Chol)或脂质体两性霉素-B(其在商标下从Gilead Sciences可商业地获得)。在本发明的优选实施方案中,脂质体是阳离子脂质体,更优选地选自二油酰磷脂酰乙醇胺(DOPE)、N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)和脂质体两性霉素-B或其组合。本发明的酿酒酵母线粒体核酸级分和脂质体可以按约1∶200至200∶1、优选1∶100至100∶1、更优选约1∶50至50∶1、甚至更优选约1∶10至10∶1、甚至更优选约1∶3至3∶1并且最优选约1∶1比率(体积/体积(v/v)和/或重量/重量(w/w))使用。
脂质体两性霉素-B是在商标(Gilead Sciences)下可商业获得的。根据本发明的优选实施方案,优选地如实施例2中所述按约1∶3至1∶1(v/v);1∶100(w/w)的比率,使用酿酒酵母线粒体核酸级分(即NA-B2级分)和
本发明的优选阳离子脂质体组合是二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)。比率1∶1(w/w)的二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)是在商标(Invitrogen,目录号18292-011或目录号18292-037)下可商业获得的。根据本发明的优选实施方案,优选地如实施例1(NA级分)和实施例2(NA-B2级分)中所述,酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)和为1∶1(v/v和/或w/w)的比率。本发明的另一个优选组合是二油酰磷脂酰乙醇胺(DOPE)、N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)和脂质体两性霉素-B。本领域技术人员能够确定本发明的酿酒酵母线粒体核酸级分、和脂质体两性霉素-B之间的哪个比率是最合适的。
如完整申请书通篇范围内所用,“亚微水包油乳液”(submicronoil-in-water emulsion)包含无毒可代谢油和商业乳化剂。无毒可代谢油包括,但不限于植物油、鱼油、动物油或合成制备的油。商业乳化剂包括,但不限于基于失水山梨糖醇的非离子表面活性剂(例如失水山梨糖醇三油酸酯或聚氧乙烯失水山梨糖醇单油酸酯)或衍生自例如月桂基、乙酰基、硬脂基和油基醇的聚氧乙烯脂肪酸醚。亚微水包油乳液广泛记录在技术人员可获得的文献中(例如WO 90/14837;TAMILVANAN S.,水包油脂质乳液:对于肠胃外和眼部送递系统的意义(Oil-in-water lipid emulsions:implications for parenteral and ocular delivering systems),Prog Lipid Res.2004年11月;43(6):489-533)。本发明的酿酒酵母线粒体核酸级分和亚微水包油乳液可以按约1∶200至200∶1、优选1∶100至100∶1、更优选约1∶50至50∶1、甚至更优选约1∶10至10∶1、甚至更优选约1∶3至3∶1并且最优选约1∶1比率(体积/体积(v/v)和/或重量/重量(w/w))使用。
如完整申请书通篇范围内所用,“微粒子”(microparticle)指从可消毒的无毒且生物可降解材料(如,但不限于聚(α-羟酸)(例如聚丙交酯或聚(D,L-丙交酯共乙交酯)))、聚羟基丁酸、聚己内酯、聚原酸酯、聚酐、聚乙烯醇和乙烯-乙酸乙烯酯)形成的直径约100nm至约150μm的粒子。微粒子广泛记录在技术人员可获得的文献中(例如RAVI KUMAR M.N.V.,作为受控grud送递装置的纳米粒子和微粒子(Nano and microparticles ascontrolled grud delivery devices),J.Pharm.Pharmaceut.Sci 3(2):234-258,2000;WO 07/084418)。本发明的酿酒酵母线粒体核酸级分和微粒子可以按约1∶200至200∶1、优选1∶100至100∶1、更优选约1∶50至50∶1、甚至更优选约1∶10至10∶1、甚至更优选约1∶3至3∶1并且最优选约1∶1比率(体积/体积(v/v)和/或重量/重量(w/w))使用。
如完整申请书通篇范围内所用,“ISCOM”指糖苷类物质如三萜皂甙(尤其QuilA)与含有疏水区的抗原之间所形成的免疫原性复合物。ISCOM广泛记录在技术人员可获得的文献中(例如BARR I.J.和GRAHAM F.M.,“ISCOM(免疫刺激复合物):前十年回顾(ISCOMs(immunostimulating complexes):The first decade”)”,Immunology andCell Biology(1996)74,8-25;WO 9206710)。本发明的酿酒酵母线粒体核酸级分和ISCOM可以按约1∶200至200∶1、优选1∶100至100∶1、更优选约1∶50至50∶1、甚至更优选约1∶10至10∶1、甚至更优选约1∶3至3∶1并且最优选约1∶1比率(体积/体积(v/v)和/或重量/重量(w/w))使用。
如完整申请书通篇范围内所用,“聚合物”包括,但不限于多聚赖氨酸、多聚精氨酸、多聚鸟氨酸、精胺和亚精胺。本发明的酿酒酵母线粒体核酸级分和聚合物可以按约1∶200至200∶1、优选1∶100至100∶1、更优选约1∶50至50∶1、甚至更优选约1∶10至10∶1、甚至更优选约1∶3至3∶1并且最优选约1∶1比率(体积/体积(v/v)和/或重量/重量(w/w))使用。
本申请人已经惊讶地发现,与单独施用本发明酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)和单独施用脂质体两性霉素-B(即)所致的应答相比,与脂质体两性霉素-B(即)组合施用的本发明酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)统计学显著地提高Th1型应答(即γ干扰素(IFN-γ)、白细胞介素-2(IL-2)和/或白细胞介素-12(IL-12)的产生),其中由单独施用本发明酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)所致的应答高于由单独施用脂质体两性霉素-B(即)所致的应答。该效应一般地称作(如完整申请书通篇范围内所用)‘协同效应(synergic effect)’或(synergistic effect)’。因组合施用NA-B2级分和脂质体两性霉素-B(即)所致的协同效应在实施例6中描述并且在图4(γ干扰素(IFN-γ))和图5(白细胞介素-12(IL-12))中显示。
就此方面而言,本发明也涉及具有协同效应的佐剂组合物,其包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;和
(ii)脂质体两性霉素-B。
就此方面而言,本发明也涉及具有协同效应的疫苗组合物,其包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;
(ii)脂质体两性霉素-B;和
(iii)抗原。
本申请人已经惊讶地发现,与由单独施用本发明酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)和单独施用二油酰磷脂酰乙醇胺(DOPE)与N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)(即)所致的应答相比,随二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)(即)组合施用的本发明酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)统计学显著地提高Th1型应答(即γ干扰素(IFN-γ)、白细胞介素-2(IL-2)和/或白细胞介素-12(IL-12)的产生),其中由单独施用本发明酿酒酵母线粒体核酸级分(即NA级分;NA-B2级分)所致的应答高于由单独施用二油酰磷脂酰乙醇胺(DOPE)与N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)(即)所致的应答。该效应一般称作(如完整申请书通篇范围内所用)‘协同效应(synergic effect)’或‘(synergistic effect)’。由组合施用NA-B2级分和二油酰磷脂酰乙醇胺(DOPE)与N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)(即)所致的协同效应在实施例6中描述并且在图5(白细胞介素-12(IL-12))中显示。
就此方面而言,本发明也涉及具有协同效应的佐剂组合物,其包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;和
(ii)二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)。
就此方面而言,本发明也涉及具有协同效应的疫苗组合物,其包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;
(ii)二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA);和
(iii)抗原。
本发明也涉及试剂盒(kit of part)。该试剂盒可以是将全部组分(即本发明的酿酒酵母线粒体核酸级分;抗原;改善酿酒酵母线粒体核酸级分和/或抗原的转染效率和/或稳定性的物质)容纳在一起的单一容器,或它可以是分别地容纳组分的单个剂量的多个容器,如泡罩包装(blister pack)。试剂盒也可以具有用于说明不同组分的施用时间的说明书。说明书将指导受试者在适宜时间服用所述组分。例如,用于送递组分的合适时间可以为症状出现时。备选地,用于施用组分的合适时间可以按常规时间表如按月或按年进行。不同的组分可以同时或分开地施用,只要它们在时间上足够接近地施用以产生协同免疫应答即可。
根据第一个优选的实施方案,试剂盒包含含有至少一种本发明酿酒酵母线粒体核酸级分的容器和含有至少一种抗原的容器,以及用于说明施用所述组分的时间的说明书。
根据另一个优选的实施方案,试剂盒包含含有至少一种本发明酿酒酵母线粒体核酸级分的容器、含有至少一种抗原的容器、含有至少一种改善酿酒酵母线粒体核酸级分和/或抗原的转染效率和/或稳定性的物质(所述的物质更优选地是脂质体两性霉素-B和/或二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA))的容器,和用于说明施用所述组分的时间的说明书。
本发明的酿酒酵母线粒体核酸级分可以用于制备药物组合物(并且更尤其是佐剂组合物和疫苗组合物),所述的药物组合物旨在对哺乳动物预防和/或治疗本领域技术人员已知的任何疾病,例如癌症、传染病、变态反应和/或自身免疫病。
术语“癌(cancer)”、“瘤(neoplasm)”、“肿瘤(tumor)”和“癌瘤(carcinoma)”在本文中可互换地用来指这样的细胞,它们显示相对自主的生长,从而表现出以细胞增殖明显失控为特征的异常生长表型。通常,本申请中待预防或治疗的细胞包括癌前的(例如良性)、恶性、转移前、转移的和非转移的细胞。“癌”(如完整申请书通篇范围内所用)包括,但不限于肺癌(例如小细胞肺癌和非小细胞肺癌)、支气管癌、食管癌、咽癌、头颈癌(例如喉癌、唇癌、鼻腔与副鼻窦癌和咽喉癌)、口腔癌(例如舌癌)、胃癌(例如stomach cancer)、肠癌、胃肠癌、结肠癌、直肠癌、结直肠癌、肛门癌、肝癌、胰腺癌、尿道癌、膀胱癌、甲状腺癌、肾癌、癌瘤、腺癌、皮肤癌(例如黑素瘤)、眼癌(例如成视网膜细胞瘤)、脑癌(例如神经胶质瘤、成神经管细胞瘤和脑星形细胞瘤)、中枢神经系统癌、淋巴瘤(例如皮肤B-细胞淋巴瘤、Burkitt淋巴瘤、霍奇金综合征和非霍奇金淋巴瘤)、骨癌、白血病、乳腺癌、生殖道癌、子宫颈癌(例如子宫颈上皮内瘤变)、子宫癌(例如子宫内膜癌)、卵巢癌、阴道癌、外阴癌、前列腺癌、睾丸癌。“癌”也指病毒诱导的肿瘤,包括但不限于乳头瘤病毒诱导的癌瘤、疱疹病毒诱导的肿瘤、EBV诱导的B细胞淋巴瘤、乙型肝炎病毒诱导的肿瘤、HTLV-1诱导的淋巴瘤和HTLV-2诱导的淋巴瘤。在本发明的优选实施方案中,如实施例5中所述,本发明的酿酒酵母线粒体核酸级分可以用于制备药物组合物,所述的药物组合物旨在用于对哺乳动物预防和/或治疗肾癌。
如完整申请书通篇范围内所用,“传染病”指由传染性生物引起的任何疾病。传染性生物包括,但不限于,病毒(例如单链RNA病毒、单链DNA病毒、人类免疫缺陷病毒(HIV)、甲、乙和丙型肝炎病毒、单纯疱疹病毒(HSV)、巨细胞病毒(CMV)、呼吸道合胞体病毒(RSV)、Epstein-Barr病毒(EBV)或人乳头瘤病毒(HPV))、寄生物(例如原生动物和后生动物病原体如疟原虫属(Plasmodia)物种、利什曼属(Leishmania)物种、血吸虫属(Schistosoma)物种或锥虫属(Trypanosoma)物种)、细菌(例如分枝杆菌,尤其结核分枝杆菌、沙门氏菌、链球菌、大肠杆菌或葡萄球菌)、真菌(例如假丝酵母属(Candida)物种或曲霉属(Aspergillus)物种)、卡氏肺囊虫(Pneumocystis carinii)和朊病毒。在本发明的优选实施方案中,如实施例4中所述,本发明的酿酒酵母线粒体核酸级分可以用于制备药物组合物,所述的药物组合物用于对哺乳动物预防和/或治疗人乳头瘤病毒(HPV)。
如完整申请书通篇范围内所用,“变态反应”指由变应原(例如本发明前面所提及的变应原)引起的任何变态反应。
如完整申请书通篇范围内所用,“自身免疫病”可以分成两个一般类型:“系统性自身免疫病”(即,损害众多器官或组织的病症)和‘局限化自身免疫病’(即,仅损害单一器官或组织的病症)。然而,“局限化自身免疫病”的影响可以因间接影响其他身体器官和系统而是系统性的。“系统性自身免疫病”包括但不限于类风湿关节炎,其可以影响关节并可能影响肺、皮肤;狼疮,包括系统性红斑狼疮(SLE),其可以影响皮肤、关节、肾脏、心脏、脑、红细胞以及其他组织和器官;硬皮病,其可以影响皮肤、肠和肺;Sjogren综合征,其可以影响唾液腺、泪腺和关节;Goodpasture综合征,其可以影响肺和肾脏;韦格纳肉芽肿,其可以影响鼻窦、肺、肾;风湿性多肌痛,其可以影响大肌肉群;以及颞动脉炎/巨细胞动脉炎,其可以影响头部和颈部的动脉。“局限化自身免疫病”包括但不限于1型糖尿病,其影响胰岛;桥本甲状腺炎和Grave病,其影响甲状腺;乳靡病(CeliacDisease)、克罗恩病和溃疡性结肠炎,其影响胃肠道;多发性硬化症(MS)和Guillain-Barre综合征,其影响中枢神经系统;Addison病,其影响肾上腺;原发性胆道硬化,硬化性胆管炎,和自身免疫性肝炎,其影响肝脏;以及雷诺现象,其可以影响手指、脚趾、鼻、耳。
包含本发明酿酒酵母线粒体核酸级分的药物组合物(并且更尤其地是佐剂组合物和疫苗组合物)还可以包含可药用载体。该可药用载体优选地是等渗的、低渗的或略微高渗的并且具有相对低的离子强度,例如蔗糖溶液。另外,这种载体可以含有任意溶剂,或水性或部分水性的液体,如无热原无菌水。另外,调节并且缓冲该药物组合物的pH,从而满足体内使用的要求。所述药物组合物(并且更尤其地是佐剂组合物和疫苗组合物)也可以包括可药用的稀释剂、助剂或赋形剂,以及增溶剂、稳定剂和防腐剂。对于注射施用,优选在水溶液、非水溶液或等渗溶液中的制剂。该制剂可以按单个剂量或按多个剂量,以液体或干燥(粉末、冻干物和其他)形式提供,所述干燥形式可以在使用时用适宜稀释剂重构(reconstituted)。
本发明也涉及使哺乳动物中的免疫应答朝向针对抗原的Th1型应答的方法,包含施用抗原和由本发明方法制备的酿酒酵母线粒体核酸级分至该哺乳动物。在一个实施方案中,该方法包括同时地施用该抗原和本发明的酿酒酵母线粒体核酸级分。备选地,该方法包括依次地施用该抗原和本发明的酿酒酵母线粒体核酸级分。术语“依次的”意指在一个时间段内相继地施用所述组分给受试者。因而,依次施用可以允许一种组分在另一种组分后数分钟或数小时范围内施用。例如,实施例5描述了本发明的酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原用于制备治疗癌症的药物组合物的用途,其中酿酒酵母线粒体核酸级分(即NA级分)晚于MUC-1抗原1小时注射。
施用本发明的药物组合物(并且更尤其地是佐剂组合物和疫苗组合物),并且更尤其地施用所述组合物的不同组分(即本发明的酿酒酵母线粒体核酸级分;抗原;改善酿酒酵母线粒体核酸级分和/或抗原的转染效率和/或稳定性的物质),可以通过熟练技术人员已知的任何手段实现。优选的施用途径包括但不限于皮内、皮下、口服、肠胃外、肌内、鼻内、肿瘤内、舌下、气管内、吸入、眼、阴道和直肠。根据优选的实施方案,皮下或皮内送递本发明的药物组合物(并且更尤其地是佐剂组合物和疫苗组合物),并且更尤其地送递所述组合物的组分。根据甚至更优选的本发明实施方案,在相同部位施用本发明的抗原和酿酒酵母线粒体核酸级分。例如,实施例5描述了本发明的酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原用于制备治疗癌症的药物组合物的用途,其中酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原在相同的部位皮下施用。
施用可以以单一剂量进行,或按某个时间间隔重复一次或多次剂量。期望地,药物组合物,并且更尤其地所述药物组合物的组分,按每周间隔施用1次至10次。例如,实施例5描述了本发明的酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原用于制备治疗癌症的药物组合物的用途,其中酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原按每周间隔施用3次。
抗原施用的剂量也是可变的,并且可以随多种参数进行调整,所述的参数尤其是施用模式;使用的药物组合物;宿主生物的年龄、健康和重量;症状的本质和程度;并行疗法的类型;治疗频率;和/或对预防或治疗的需求。进一步微调计算结果以确定适宜的治疗剂量可以由执业医生在考虑相关情况下常规地进行。
作为一般指导,包含MVA的组合物的合适剂量从约104至1010pfu(蚀斑形成单位),有利地约105至108pfu;包含腺病毒的组合物从约105至1013iu(感染单位),有利地约107至1012iu。基于载体质粒的组合物可以按10μg至20mg、有利地100μg至2mg的剂量施用。在本发明的优选实施方案中,药物组合物以包含5×105pfu至5×107pfu MVA载体的剂量施用。例如,实施例5描述了本发明的酿酒酵母线粒体核酸级分(即NA级分)和MUC-1抗原用于制备治疗癌症的药物组合物的用途,其中包含于MVA载体中的MUC-1抗原以5×107pfu施用。
当本发明的用途、方法、佐剂组合物、疫苗组合物或试剂盒用于治疗癌症时,本发明的用途、方法、佐剂组合物、疫苗组合物或试剂盒可以与一种或多种常规治疗方法(例如放疗法、化疗法和/或手术)联合实施。多种治疗手段的使用为患者提供更宽的介入。在一个实施方案中,本发明方法可以在手术介入之前或之后。在另一个实施方案中,它可以在放疗(例如,γ放疗)之前或之后。本领域技术人员可以容易地配置可以使用的适宜放疗方案和参数(例如,见PEREZ,放射肿瘤学原理与实践(Principles andpractice of radiation oncology).第2版LIPPINCOTT,1992;如本领域技术人员易于明了的,使用适宜的调整和改动)。
本发明还涉及用于改善对癌症患者的治疗的方法,所述的癌症患者正在用化疗药进行化疗,所述方法包括用如上所公开的方法一起共同治疗该患者。
本发明还涉及用于改善细胞毒性药物或放疗的细胞毒性效力的方法,所述方法包括对需要该治疗的患者,联合如上所公开的方法进行共同治疗。
当本发明的用途、方法、佐剂组合物、疫苗组合物或试剂盒用于治疗传染病时,本发明的用途、方法、佐剂组合物、疫苗组合物或试剂盒可以随其他治疗性化合物如抗生素、抗真菌化合物、抗寄生物化合物和/或抗病毒化合物的应用一起实施。
本发明还涉及用于改善抗生素、抗真菌药物、抗寄生物药物和/或抗病毒药物的治疗效力的方法,所述方法包括对需要该治疗的患者,联合如上所公开的方法进行共同治疗。
在另一个实施方案中,本发明的用途、方法、佐剂组合物、疫苗组合物或试剂盒按照初免-加强免疫(prime-boost)治疗模式实施,其包括相继地施用一种或多种初免组合物和一种或多种加强免疫组合物。一般,初免组合物和加强组合物使用包含或编码至少一个共同抗原结构域的不同载体(vehicle)。初免组合物首先施用至宿主生物并且加强组合物相继地在1日至12个月的时间之后施用至相同的宿主生物。本发明的方法可以包括初免组合物的1至10个依次施用,后续加强组合物的1至10个依次施用。可能期望地,注射间隔期是大约1周至6个月。另外,可以在相同部位或在不同部位,通过相同的施用途径或通过不同的施用途径,施用初免组合物和加强组合物。
附图简述
图1:在1xTAE(Tris-乙酸盐-EDTA)缓冲液中在琼脂糖凝胶(1%)内的NA级分、NA-B1级分和NA-B2级分,伴有或不伴有RNA酶处理。
图2:由皮下注射(第0日;第7日和第14日)HPV16E7抗原(10μg)与NA级分(25μg)或NA-B2级分(0.4μg)所产生的体内ELISpot γ干扰素(IFN-γ)。
图3:皮下施用(在第4日、第11日和第18日)5×107pfu表达MUC1抗原和hIL-2的MVA株(MVA9931)和(1小时后)NA级分(50μg)对B6D2小鼠的肿瘤体积的影响,其中所述的B6D2小鼠皮下注射了3×105个RenCa-MUC-1细胞(在第1日)。肿瘤内(I.T.)施用(在第4日、第11日和第18日)(50μg+50μg)的影响。一周2次测量肿瘤体积。
图4:在用NA-B2级分(0.4μg或1.2μg)、(120μg)或(0.4μg+120μg或1.2μg+120μg)处理的人未成熟的单核细胞衍生的树突细胞(moDC)中γ干扰素(IFN-γ)的诱导。
图5:在用NA-B2级分(0.2μg)、(10μg)、(80μg,120μg或160μg)、(0.2μg+10μg)和(0.2μg+120μg)处理的人未成熟moDC中白细胞介素-12(IL-12)的诱导。
实施例
为举例说明本发明,提供以下实施例。所述实施例不意图以任何方式限制本发明的范围。
实施例1:酿酒酵母线粒体核酸级分(NA级分)的制备
将等份样的冷冻酿酒酵母(S.c.)AH109(Clonetech)铺展在由1%酵母提取物,1%Bacto胨、2%葡萄糖、2%琼脂(BD Sciences)和100μg/ml腺嘌呤(Fluka 01830-5G)组成的YPG平板上。在28℃至30℃培育2日,用刮勺取得等份样的酿酒酵母S.c.AH109以接种倾入一只500ml瓶中的100ml液体YPG/腺嘌呤培养基。在搅拌下(200转/分钟)28℃过夜孵育后,分别地将15ml这种预培养物转移到含有500ml YPG/腺嘌呤培养基的6只2000ml瓶中。这些培养物(总计3升)在搅拌下(200转/分钟)28℃孵育过夜。在600nm处测量的光密度(OD600)为2+/-0.5时,将培养物以3500转/分钟(Sorvall离心机,500ml管)于4℃离心15分钟。
细胞沉淀用蒸馏水洗涤一次,例如每份从3升培养物衍生的沉淀用1升蒸馏水。在离心(Sorvall,3500转/分钟,在4℃持续15分钟)后,将细胞沉淀溶解于PBS中,使所得悬液的OD600是约100(例如从3升培养物衍生的细胞沉淀溶解于40ml PBS中)。从该步骤起,样品总是维持在冷温(4℃):将30ml所述细胞悬液转移到125ml聚对苯二甲酸乙二酯(PETG)瓶中并且与30ml无菌玻璃珠(直径0.7mm)混合。将混合物以最大速度持续1分钟涡旋并交替以冰上孵育1分钟,进行5次涡旋(桌面涡旋混合仪TOPMIX 94323 BIOBLOCK Scientifique)。细胞裂解物使用借助1000μl蓝色吸头延长以避免抽吸玻璃珠的5ml玻璃吸管回收,并且连同用来洗涤玻璃珠的10ml PBS一起转移到50ml离心管(Corning)中。
将细胞裂解物在4000转/分钟于4℃(Sorvall)离心10分钟以使膜残片以及细胞核沉淀。
获得的上清液在12ml管中以39000转/分钟在4℃于SW40转头(105000g)中超速离心90分钟,以沉淀线粒体。沉淀溶解于冷的PBS中(例如,从初始9升酿酒酵母培养物获得的沉淀溶解于100ml PBS中)。所得的线粒体级分命名为SN。
获得的SN级分用酚处理以从蛋白质和脂质中提取核酸。为此,将等体积的Tris缓冲的酚(Amresco)添加至该悬液,以最大速度在室温(RT)涡旋混合1分钟并且离心(例如,50ml Falcon管在室温于Hareus离心机中以5000转/分钟离心10分钟)。将上层水相分离并转移在一只新管中。酚提取过程重复三次。3次酚提取后回收的上层水相随后用二氯甲烷(p.A.;Merck)提取两次:添加等体积的二氯甲烷并且将混合物在室温涡旋混合30秒并离心(例如,50ml Falcon管在室温于Hareus离心机中以5000转/分钟离心10分钟)。回收水相并且重复二氯甲烷处理。
通过乙醇沉淀法从分离的上清液回收核酸:以上清液体积的1/10添加3M乙酸钠pH 5以及添加2体积的乙醇(无水乙醇)。在4℃过夜孵育后,将溶液离心(例如,50ml Falcon管在4℃于Hareus离心机中20分钟)。沉淀用冷的70%乙醇洗涤。在完全干燥之后,沉淀溶解于TE pH7.5中(例如从d)步骤中获得的100ml悬液中衍生的沉淀溶解于20-25ml TE pH7.5中,形成如由260nm处的光密度所度量的约1μg/μl核酸浓度)。所得的线粒体核酸级分命名为NA级分。
已经根据所述方法进行了从9升酿酒酵母培养物开始的3次独立大规模酿酒酵母核酸级分(即NA级分)制备。这3个制备物导致相当的特征。全部3个制备物中由LAL测定法测量的内毒素水平是低的且相当的(在0.5和0.7EU/ml之间)。
也已经进行从酿酒酵母W303(Biochem)开始的酿酒酵母核酸级分(即NA级分)制备。
实施例2:从NA级分(NA-B2级分)分离线粒体RNA
在1xTAE(Tris-乙酸盐-EDTA)缓冲液中在1%琼脂糖凝胶上跑根据实施例1中所述方法制备的NA级分。
图1中所示结果显示与DNA标记λ-HindIII/PhiX174-HaeIII(在图1中叫做M)相比,可以清楚地区分3组核酸:
(1)一条迁移在20Kbp附近的清晰条带,叫做NA-B1级分;
(2)一条迁移在4Kbp附近的清晰条带,叫做NA-B2级分;和
(3)在1000和约100bp之间迁移的弥散分子,叫做NA小级分。
随后,使用轻微UV和手术刀片从琼脂糖凝胶切下各条带或条带群,以进行NA-B1级分,NA-B2级分和NA小级分的纯化。将切下的琼脂糖块转移到一个“双管结构体”(=0.5ml管插在切下盖的2ml管中,其中所述的0.5ml管在底部带有通过施加热针而产生的孔,孔中塞有棉花充当过滤器)中,在低于-60℃冷冻,在室温于台面离心机中以5000转/分钟离心15分钟(直至材料彻底融化),随后以14000转/分钟离心2分钟。在下部管中回收的溶液转移到新管中。如实施例1中所述使用乙酸钠和乙醇沉淀核酸。沉淀溶解于TE pH7.5中。一般,从琼脂糖凝胶上的3mg NA级分开始,回收约8μg的NA-B2级分,通常在TE pH7.5中溶解至20ng/μl浓度。
伴有或不伴有RNA酶处理(100mg/ml;Qiagen),NA级分、NA-B1级分和NA-B2级分随后在1xTAE(Tris-乙酸盐-EDTA)缓冲液中在1%琼脂糖凝胶上跑胶。
如图1中所示的结果表明:
(1)NA-B1级分证明是HaeIII-敏感且RNA酶A不敏感的,说明NA-B1级分是DNA;
(2)NA-B2级分和NA小级分是HaeIII不敏感且RNA酶A敏感的,说明这些分子是RNA。
相同结果已经用从酿酒酵母AH109(Clonetech)得到的级分和从酿酒酵母W303(Biochem)得到的级分获得。
为产生NA-B2级分(其将在以下实施例中测试),将NA-B2级分(20ng/μl)与(1μg/μl;Invitrogen,目录号18292-011或目录号18292-037)以比率1∶1(v∶v)混合。
实施例3:NA级分和NA-B2级分刺激人Toll样受体(TLR)的能力
细胞:人胚肾细胞293(HEK)用允许组成型表达1种或2种人源Toll样受体(hTLR)的质粒稳定转染。产生的细胞系293/hTLR2-CD14、293/hTLR3、293/hTLR4-MD2-CD14、293/hTLR5、293/hTLR2/6、293/hTLR7、293/hTLR8和293/hTLR9购自InvivoGen(San Diego、CA、美国)。全部细胞系均在杀稻瘟素S(10μg/ml,InvivoGen)存在下于补充有10%胎牛血清、40μg/ml庆大霉素、2mM谷氨酰胺、1mM丙酮酸钠(Sigma)和1x非必需氨基酸(NEAA,Gibco)的Dulbecco极限Eagle培养基(DMEM)中培养。在293/hTLR2-CD14和293/hTLR4-MD2-CD14的情况下,添加潮霉素B至100μg/ml浓度。全部8个细胞系用NF-kB诱导型报道质粒pNiFty(InvivoGen)稳定转染。pNiFty编码在工程化ELAM1启动子控制下的萤火虫萤光素酶基因,其中所述的工程化ELAM1启动子组合5个NF-kB位点和近端ELAM启动子。在100μg/ml Zeocin(InvivoGen)存在下选择稳定的转染子。出现的克隆“hTLRx-luc”就相对于相应对照TLR配体的EC50和倍数诱导进行了表征。选择具有最低EC50和高倍数诱导及良好/可接受生长行为的克隆。在表1中列出所保留的克隆和它们的特征。
表1:
对照细胞系293-luc-2-8(293-luc):HEK-293细胞用NF-kB诱导型报道质粒pNiFty2稳定转染。在100μg/ml Zeocin(InvivoGen)存在下选择稳定的转染子。对阳性克隆293-luc-2亚克隆,留下克隆293-luc-2-8。产生这个对照细胞系以作为NF-kB途径的TLR非依赖性刺激的对照。
RT-PCR实验已经显示(数据未显示)全部细胞系对于RLH家族成员rig-I(维甲酸诱导型基因1)和mda-5(黑素瘤分化抗原5)信使均是阳性(KR08001p75,Ren ée Brandely,2008年10月)。此外,显示(数据未显示)全部细胞系可以受被供应商描述为MDA-5配体的制剂poly(I:C)“polyICLyoVec”(InvivoGen)刺激。该结果说明了MDA-5在全部细胞系(TLR和对照细胞系)中的功能性。
体外TLR试验--方法:在96孔平板中铺种稀释于补充有2%胎牛血清、40μg/ml庆大霉素、2mM谷氨酰胺、1mM丙酮酸钠(Sigma)和1xMEM非必需氨基酸(NEAA,Gibco)的DMEM中的细胞。次日,单独或与组合(如实施例1中所述)的NA级分(母液:1mg/ml)以浓度16μg/ml以及其3倍连续稀释物进行添加。作为阳性对照,细胞系用规定量的其相应参考配体刺激。在刺激(18-20小时)后当日,在含有125mM TrispH 7.8、10mM EDTA、5mM DTT和5%Triton X-100的100μl缓冲液中裂解细胞。在添加50μl萤光素酶发光缓冲液(1x萤光素酶发光缓冲液:20mM Tris pH7.8,1.07mM MgCl2,2.7mM MgSO4,0.1mM EDTA,33.3mM DTT,470μM萤光素,530μM ATP和270μM辅酶A)后,10μl裂解物中的萤火虫萤光素酶活性通过累计测量1秒内闪光(LB96PMicrolumat,Berthold)而定量。所得的相对光单位(RLU)表述为与对照配体相比的诱导百分数并使用S型剂量反应方程式以软件Graph Pad Prism 4分析(测定EC50)。
体外TLR试验NO 1:根据前述方法,在TLR细胞系和对照细胞系上测试单独或与组合的两个独立批次的NA级分(批次1:0.6EU/ml;批次2:0.77EU/ml)。在表2中显示以相对于相应对照配体(见表1)所观察到的百分数表述的最大活化作用。
表2:
表2中所示的结果表明:
(1)用NA级分在hTLR 3、4和7中观察到刺激作用(批次1以及批次2);
体外TLR试验NO 2:根据前述方法,在TLR细胞系和对照细胞系上测试在添加之前用RNA酶(100mg/ml;Qiagen)处理的NA-B1级分、NA-B2级分(1.3EU/ml)和NA级分(0.7EU/ml)。表3中描述结果。
表3:
如表3中所示的结果表明:
就HEK-293-TLR细胞系刺激作用而言,采用来自AH119(Clonetech)的NA-B2所获得的结果是与采用来自酿酒酵母株W303(Biochem)的NA-B2所获得的结果相当的。
实施例4:NA级分或NA-B2级分和HPV16E7抗原用于制备使免疫应答朝向针对HPV16E7抗原的Th1型应答的药物组合物的用途
动物模型:SPF健康雌性C57BL/6小鼠从Charles River(Les Oncins,法国)获得。抵达时,动物是6周龄。在实验开始时,它们是7周龄。动物保持在给予空调以提供每小时最少11次空气交换的单个专有房间中。温度和相对湿度范围分别在20℃和24℃以及40%至70%范围内。自动控制光照以产生12小时白昼和12小时黑暗的周期。通过定期检验岗哨动物对照,检查无特定病原体状态。整个本研究期间,动物自由获取RM1型无菌膳食(Dietex France,Saint Gratien)。通过瓶子自由获取无菌水。
体内ELISpot γ干扰素(IFN-γ):
IFN-γELIspot测定法是一项功能性试验,确定体内初免的T细胞在用特定肽体外再刺激后分泌IFN-γ的能力。
动物按1周间隔(第0日;第7日和第14日)用如表4中所述的制备物(在尾基部)皮下注射3次。
表4:
随后在最后注射后7日处死动物,并且它们的脾细胞用来确定再刺激时分泌IFN-γ的R9F特异性CD8+T细胞的频率。
ELISpot平板用2.5μg/ml稀释于无菌DPBS中的大鼠抗小鼠IFN-γ单克隆抗体(100μl/孔;BD Pharmingen,编号:551216)包被。该平板随后被覆盖并在室温孵育过夜或在37℃孵育4小时或在4℃孵育24小时。随后进行5次无菌PBS(200μl/孔)洗涤。平板随后用200μl/孔完全培养基在37℃封闭1小时。
为制备用于该试验的淋巴细胞,6孔平板中每孔放入5ml完全培养基(RPMI;FBS 10%;40μg/ml庆大霉素;2mM谷氨酰胺;5x10-5M β-巯基乙醇)。在6-孔培养平板的孔中在细胞滤网(BD Bioscience;编号352360)上汇集来自相同小鼠组的脾脏。用注射器柱塞压碎脾脏并抛弃细胞滤网。脾细胞用5ml完全培养基收集并随后转移到冰上的15ml falcon管中。随后进行在400×g和在室温(22℃)为期3分钟的离心。细胞在室温重悬于8ml完全培养基中。将8ml淋巴细胞或脾细胞悬液铺展在4ml淋巴细胞分离细胞介质(TEBU BIO,编号:CL5031)上。随后进行在1500×g和在室温(22℃)为期20分钟的离心。将淋巴细胞收集、环出(ringed)并且用10mlRPMI极限培养基洗涤3次。在每个洗涤步骤之间进行离心(以400×g为期3分钟)并且抛弃上清液。淋巴细胞随后重悬于2ml的RBC裂解缓冲液(BD Pharmingen;编号555899)中。添加裂解液后立即温和地涡旋混合每只管,然后在室温孵育15分钟。随后进行以400×g为期3分钟的离心并且抛弃上清液。细胞用10ml完全培养基洗涤并且随后以400×g离心3分钟。抛弃上清液。在细胞于6ml完全培养基中(取决于沉淀的大小)重悬后,在Malassez小室上计数细胞,并且调整细胞浓度为完全培养基中每毫升1×107个细胞。
ELISpot测定法本身如下进行:在伴有或没有2-4μg/ml目的肽(即HPV16E7肽抗原)的情况下每孔添加100μl完全培养基。添加100μl细胞悬液。在37℃于5%CO2中孵育20h后,采用H2O洗涤缓冲液(PBS,1%PBS)进行2个洗涤步骤,随后在PBS洗涤缓冲液中进行5个洗涤步骤(拍干)。生物素化的大鼠抗小鼠IFN-γ单克隆抗体(BD Pharmingen,编号:554410)以4μg/ml稀释于抗体混合缓冲液中并且100μl/孔分配。该平板在室温于黑暗下孵育2小时。进行在PBS洗涤缓冲液(PBS,0.05%Tween 20)中的5个洗涤步骤(拍干)。链霉抗生物素-碱性磷酸酶随后(1/1000)稀释于抗体混合缓冲液中。添加100μl/孔并且在室温于黑暗下孵育1小时。随后进行在PBS洗涤缓冲液中的5个洗涤步骤,然后是用PBS的2个洗涤步骤(拍干)。随后添加100μl/孔的BCIP/NBT(SIGMA;编号B5655)并且将其在室温孵育直至蓝色点显现(最多2分钟)。用水彻底清洗(拍干)后,用ELISpot读数仪进行ELISpot平板的分析。在每孔上进行目视质量控制(比较扫描结果和平板)以确保计算机给出的计数匹配画面的真实性(排除潜在的人为现象)。将原始数据转换成直方图。结果表述为一式三份的每1×106个淋巴细胞的点形成单位数(sfu)(均数)。使用未再刺激的孔,使用式:[均数(未再刺激的孔)]+[2×SD(未再刺激的孔)],已经测定截断值。非特异性背景的水平通过利用无关I8L肽(HPV16E1)进行再刺激而揭示。
图2中所示的结果表明:
(1)用HPV16E7注射后不存在分泌IFN-γ的R9F特异性(HPV16E7蛋白)T细胞;
(2)向HPV16E7蛋白质添加NA级分(25μg)或NA-B2级分(0.4μg)后分泌IFN-γ的R9F特异性细胞的水平是显著的。NA级分和NA-B2级分具有佐剂能力,该能力特异性引起当再刺激时能够分泌Th1细胞因子IFN-γ的循环CD8+T细胞的频率提高。
实施例5:NA级分和MUC-1抗原用于制备治疗癌症的药物组合物的用途
每种载体构建体的名称和简述(见表5)
表5
使用的动物模型是如实施例3中所述的SPF健康雌性B6D2小鼠。
RenCa-MUC-1肿瘤细胞:RenCa是实验性鼠肾癌模型(ChakrabartyA.等人.AntiCancer Res.1994;14:373-378;Salup R.等人.Cancer Res 198646:3358-3363)。在转染表达MUC-1肽的质粒后,获得RenCa-MUC-1细胞。此类细胞在它们的表面表达MUC1抗原。RenCa-MUC-1细胞在含有10%灭活胎牛血清、2mM L-谷氨酰胺、0.04g/l庆大霉素和0.6mg/ml潮霉素的DMEM中培养。
免疫:对于免疫治疗实验,B6D2雌性小鼠在第1日于右侧腹用3×105个RenCa-MUC-1细胞作皮下攻击。诸小鼠在第4日、第11日和第18日用单独的溶媒(缓冲液)、5×107pfu无效MVA(MVAN33)、NA级分(50μg)、(50μg;Invitrogen,目录号18292-011或目录号18292-037)、(50μg+50μg)、单独或与NA级分(50μg)或(50μg+50μg)组合的表达MUC1和hIL-2的MVA株(MVA9931)5×107pfu(每组13只小鼠),皮下处理3次。诸小鼠还在第4日、第11日和第18日用单独的(50μg+50μg)肿瘤内处理3次。注射方案:首先注射MVA9931;1小时后,在相同部位注射NA级分或监测小鼠的存活。也使用卡尺一周两次监测肿瘤体积。当小鼠的肿瘤大小超过25mm直径时,出于伦理原因,将小鼠安乐死。
统计学:通过使用Stastistica 7.1软件(StatSoft,Inc.)的对数秩检验,分析Kaplan-Meier存活曲线,并且进行特定配对比较(specific pairwisecomparison)。P<0.05视为统计显著。
如图3中所示的结果表明,与未处理的对照相比,与NA级分(50μg)或(50μg+50μg)组合的MVATG9931对第20日(p:0.007752)和第25日(p:0.023046)的肿瘤生长产生统计显著的影响。
细胞培养:来自健康志愿者的分选的人单核细胞从EtablissementFrancais du Sang-Alsace(EFS)获得。取冷冻的细胞以浓度1x106个细胞/ml在补充有10%灭活胎牛血清、40μg/ml庆大霉素(Sigma)、2mM L-谷氨酰胺(Sigma)、1mM丙酮酸钠(Sigma,S8636)和1x非必需氨基酸(MEMNEAA,GIBCO)的RPMI(Gibco)中培养。为诱导分选的单核细胞分化成树突细胞(moDC),添加细胞因子GM-CSF(20ng/ml)和IL-4(10ng/ml)(Peprotech)。3日后,将细胞计数、离心并以密度1×106个细胞加入新鲜含补充物的培养基中。将2x106个细胞铺种在12孔平板(2ml/孔)中。再过2至3日后,如下文所述感染和/或刺激被视为未成熟moDC的细胞。
刺激:添加NA-B2级分、(Invitrogen,目录号18292-011或目录号18292-037)和(Gilead Sciences)至moDC。16-20h后,将细胞离心,上清液贮存在-20℃并由ELISA分析。
ELISA检测细胞因子:在16-20小时刺激后,使用来自Bender MedSystem的市售ELISA试剂盒测定细胞因子产生的量(IFN γ、IL12(p70)和IFN α)。ELISA测定根据制造商的手册进行。通过使用已知量的重组细胞因子获得的标准曲线,确定细胞因子的浓度。
结果:
γ干扰素(IFN-γ):如图4中所示,γ干扰素表达由单独的NA-B2级分(0.4μg或1.2μg)以及由单独的(120μg)诱导;但是通过用1.2μg NA-B2级分处理人未成熟moDC所获得的γ干扰素表达水平高于通过用120μg处理人未成熟moDC所获得的γ干扰素表达水平。另外,一起添加时,NA-B2级分和(0.4μg+120μg或1.2μg+120μg)以协同方式增加γ干扰素表达。
白细胞介素12(IL-12):如图5中所示,用0.2μg NA-B2级分处理的人未成熟moDC略微产生IL-12,而用10μg或用80μg、120μg或160μg处理的人未成熟moDC不分泌IL-12。添加至人未成熟moDC的组合(0.2μg+10μg)和组合(0.2μg+120μg)明显地刺激IL-12的分泌(协同效应)。
在以上说明书中提到的全部文献(例如专利、专利申请、出版物))在本文中通过引用的方式并入。本发明方法的多种修改和变化对于本领域技术人员将是显而易见的,而不脱离本发明的范围和精神。虽然已经联系具体的优选实施方案描述本发明,应当理解本发明不应不当地受限于此类具体的实施方案。实际上,对本领域技术人员显而易见的所描述的本发明实施方式的多种变型应当落入以下权利要求的范围内。
Claims (20)
1.酿酒酵母线粒体核酸级分和抗原用于制备使免疫应答朝向针对所述抗原的Th1型应答的药物组合物的用途,特征在于所述的酿酒酵母线粒体核酸级分由包括以下步骤的方法制备:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分。
2.根据权利要求1所述的用途,其中核酸是核糖核酸(RNA)。
3.根据权利要求1所述的用途,其中抗原选自由肿瘤相关抗原(TAA)、传染性生物特异性抗原和变应原特异性抗原组成的组。
4.根据权利要求1所述的用途,其中抗原选自由肽、核酸、脂质、脂肽和糖组成的组。
5.根据权利要求3所述的用途,其中TAA是MUC-1。
6.根据权利要求3所述的用途,其中传染性生物特异性抗原是人乳头瘤病毒(HPV)特异性抗原,优选地是HPV-16或/和HPV-18特异性抗原,并且更优选地是选自由HPV-16或/和HPV-18的E6早期编码区、HPV-16或/和HPV-18的E7早期编码区和其部分或组合组成的组中的抗原。
7.根据权利要求1所述的用途,其中抗原被包含于载体中,所述载体优选地选自质粒或病毒载体。
8.根据权利要求7所述的用途,其中病毒载体从痘病毒、优选地从痘苗病毒并且更优选地从改良安卡拉痘苗病毒(MVA)或其衍生物获得。
9.根据权利要求7所述的用途,其中病毒载体从腺病毒、腺病毒相关病毒、逆转录病毒、疱疹病毒、甲病毒或泡沫病毒或其衍生物获得。
10.根据权利要求7所述的用途,其中载体还包含在抗原是核酸时表达该抗原所必需的元件。
11.根据权利要求1所述的用途,其中药物组合物还包含一种或多种改善酿酒酵母线粒体核酸级分和/或抗原的转染效率和/或稳定性的物质,所述物质优选地选自由脂质、脂质体、亚微水包油乳液、微粒子、ISCOM和聚合物组成的组。
12.根据权利要求11所述的用途,其中脂质体是阳离子脂质体,优选地选自二油酰磷脂酰乙醇胺(DOPE)、N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)和脂质体两性霉素-B或其组合。
13.根据权利要求12所述的用途,其中所述组合是二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)。
14.根据权利要求1所述的用途,用于制备预防和/或治疗癌症、传染病、变态反应和/或自身免疫病的药物组合物。
15.具有协同效应的佐剂组合物,包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;和
(ii)脂质体两性霉素-B
16.具有协同效应的疫苗组合物,包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;
(ii)脂质体两性霉素-B;和
(iii)抗原。
17.具有协同效应的佐剂组合物,包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;和
(ii)二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA)。
18.具有协同效应的疫苗组合物,包含:
(i)由包括以下步骤的方法制备的酿酒酵母线粒体核酸级分:
a)在允许其生长的培养基中培养酿酒酵母,随后离心所述培养物;
b)破碎a)步骤中获得的酿酒酵母沉淀;
c)离心b)步骤中获得的混合物;
d)超速离心c)步骤中获得的上清液;
e)从d)步骤中获得的沉淀提取核酸;
f)从e)步骤中获得的上清液回收核酸级分;
(ii)二油酰磷脂酰乙醇胺(DOPE)和N-[1-(2,3-二油酰氧)丙基]-N,N,N-三甲基氯化铵(DOTMA);和
(iii)抗原。
19.试剂盒,其包含含有至少一种酿酒酵母线粒体核酸级分的容器和含有至少一种抗原的容器,和用于说明施用所述组分的时间的说明书。
20.试剂盒,其包含含有至少一种酿酒酵母线粒体核酸级分的容器、含有至少一种抗原的容器,包含至少一种改善酿酒酵母线粒体核酸级分和/或抗原的转染效率和/或稳定性的物质的容器,和用于说明施用所述组分的时间的说明书。
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