CN107614515B - 用于抗hiv(人免疫缺陷病毒)疗法和/或疫苗的t20构建体 - Google Patents
用于抗hiv(人免疫缺陷病毒)疗法和/或疫苗的t20构建体 Download PDFInfo
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- CN107614515B CN107614515B CN201680028387.9A CN201680028387A CN107614515B CN 107614515 B CN107614515 B CN 107614515B CN 201680028387 A CN201680028387 A CN 201680028387A CN 107614515 B CN107614515 B CN 107614515B
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Abstract
本发明涉及用于使用诸如以SEQ ID NO:1或SEQ ID NO:3显示的T20表达载体治疗HIV感染的方法和组合物。所述T20表达载体可用于多种治疗应用中,诸如离体转染树突细胞以诱导对HIV的宿主免疫应答,在基因治疗方法中进行体内局部转染以提供T20的较长期递送,或T20肽的体外产生。T20可被分泌至循环中以充当HIV感染的融合抑制剂,或可诱导对HIV或受HIV感染细胞的内源性免疫应答。或者,可将DDD肽并入包含T20或另一抗原性蛋白质或肽的融合蛋白中以增强对所述蛋白质或肽的免疫应答。
Description
相关申请
本申请根据35 U.S.C.119(e)要求2015年5月28日提交的美国临时专利申请62/167,404的权益,所述美国临时专利申请的正文以引用的方式整体并入本文。
序列表
本申请含有已通过EFS-Web以ASCII格式提交,并且据此以引用的方式整体并入本文的序列表。所述ASCII拷贝于2016年5月19日创建,名为IMM360WO1_SL.txt,并且大小是14,349字节。
发明背景
发明领域
本发明涉及用于防治性或感染后治疗人免疫缺陷病毒(HIV)的方法和组合物。组合物和方法涉及T20小环或包含T20小环的疫苗。提供一种新型T20小环构建体,其包含SEQID NO:1的核酸序列。T20小环可用于在受感染宿主中进行的抗HIV疗法(例如恩夫韦地(enfuvirtide))、基因疗法或病毒根除,或用于产生抗HIV疫苗或达成未受感染宿主中的疫苗增强。T20可单独或与一种或多种如下讨论的抗HIV剂组合使用。在一些实施方案中,T20可被表达成未缀合肽。或者,可使用包含连接于另一治疗性肽的T20的融合蛋白或肽。所有所述肽产物都可由如本文公开的小环表达,或小环自身可用于治疗目的。恩夫韦地已单独或与其它抗病毒剂组合用于在具有多药物抗性HIV的患者中进行的补救疗法。在某些实施方案中,佐剂可用于增加对T20或其它抗原的免疫应答。一种示例性抗原是如本文公开的DDD(二聚化和对接结构域)肽。
相关技术的描述
小环是附加型DNA载体,其以小(约4kb)环状表达盒形式产生,由质粒获得,但缺乏任何原核DNA(参见例如维基百科(Wikipedia)“小环(Minicircle)”)。小环已作为转基因载体用于对哺乳动物细胞的遗传修饰(同前)。它们的较小分子尺寸使得能够更高效转染,并且相较于仅持续数天起作用的标准质粒载体,历经数周时期提供持续表达(参见例如Kay等,2010,Nature Biotech 28:1287-89)。因此,小环可用于直接转染宿主细胞,或可用于产生克隆肽,其转而可用作治疗剂和/或用于疫苗接种。因为小环不含有细菌DNA序列,所以它们由宿主免疫系统识别并破坏的可能性较小(Argyros等,2011,J Mol Med(Berlin)89:515-29)。因此,它们比标准表达系统更适于体内使用,尽管体外使用的性能也得以改进(Argyros等,2011,J Mol Med(Berlin)89:515-29)。
小环产生通常涉及产生“亲本质粒”以及诱导位点特异性重组酶的活性以切除原核载体序列(Chen等,2003,Mol Ther 8:495-500)。所得小环可通过多种技术来回收。早期形式的小环缺乏复制起点,因此当发生细胞分裂时被丢失。最近,已设计包含S/MAR(骨架/基质附着区)元件的自我复制性小环(例如Argyros等,2011,J Mol Med 89:515-29)。自我复制性小环可使得由经转染细胞进行延长肽表达。存在对供更有效治疗使用的改进小环载体的需要。
T20(恩夫韦地)是被核准供人治疗使用的首个HIV融合抑制剂(Morris和Kraus,2005,J Pediatr Pharmacol Ther 10:215-470)。HIV-1感染由gp120包膜蛋白结合它的CD4细胞表面受体和共受体(CXCR4或CCR5)引发(Martinez-Munoz等,2014,Proc.Natl AcadSci USA 111:E1960-69)。gp120结合继之以所缔合的病毒跨膜gp41亚基的释放和构象变化,所述亚基为与宿主细胞进行膜融合所需(Cai等,2011,Curr Top Med Chem 11:2959-84)。恩夫韦地通过靶向gp41的构象转变,从而防止产生病毒衣壳的进入孔隙来起作用(Cai等,2011,Curr Top Med Chem 11:2959-84)。因为恩夫韦地是一种具有不良口服利用度的肽,所以它通常在由患者复原之后通过皮下注射来施用。由于治疗的长期特性,所以施用困难造成患者顺应性不良(Horne等,2009,AIDS Res Ther 6:2)。存在对将使得T20施用手段得以改进的更好T20载体的需要。
发明概述
本发明通过提供编码T20HIV融合抑制剂的产生的T20小环(MC)表达载体来满足本领域中的未解决需要。示例性T20小环DNA序列以SEQ ID NO:1显示。T20的氨基酸序列以SEQID NO:2提供。相较于包含大量原核核酸序列的传统表达载体,MC构建体展现若干优势,诸如免疫原性降低,诱导宿主免疫应答的潜力较低,构建体在宿主细胞中的稳定性较大,T20肽的表达延长以及产量增加。
T20MC构建体适用于预防HIV感染,例如作为疫苗的组分或疫苗辅助剂,或用于治疗现有HIV感染,例如通过施用所表达的T20肽或通过将T20MC表达载体并入宿主细胞中以达成体内蛋白质表达。
T20肽或T20MC载体可单独或与一种或多种选自由以下组成的组的其它已知抗HIV剂组合使用:融合抑制剂(例如恩夫韦地、马拉韦罗(maraviroc))、整合酶抑制剂(例如雷特格韦(raltegravir)、埃替格韦(elvitegravir)、度鲁特韦(dolutegravir))、逆转录酶抑制剂(例如KRV2110、齐多夫定(zidovudine)、阿巴卡韦(abacavir)、恩曲他滨(emtricitabine)、替诺福韦(tenofovir)、依曲韦林(etravirine)、利匹韦林(rilpivirine)、地达诺新(didanosine)、扎西他滨(zalcitabine)、拉米夫定(lamivudine)、司他呋啶(stavudine)、奈韦拉平(nevirapine)、依法韦仑(efavirenz))、蛋白酶抑制剂(例如沙奎那维(saqujinavir)、茚地那韦(indinavir)、利托那韦(ritonavir)、洛匹那韦(lopinavir)、奈非那韦(nelfinavir)、安普那韦(amprenavir)、地瑞那韦(darunavir)、阿扎那韦(atazanavir))和抗HIV抗体或其它结合分子(例如P4/D10、2G12、2F5、4E10、朱顶红杂合凝集素(Hippeastrum hybrid agglutinin,HHA))。已报道与T20(恩夫韦地)的组合疗法在多达90至95%的治疗患者中使病毒载量降低至低于可检测水平(参见例如McGillick等,2010,Biochem 49:3575-92)。通常承认的是组合疗法比单一药剂疗法在控制HIV感染以及预防药物抗性HIV的形成方面更有效(Jenabian等,2009,J AntimicrobChemother 64:1192-95)。许多抗HIV治疗剂在本领域中是已知的,并且可使用任何所述已知药剂。
使用所述组合疗法可阻断或预防HIV感染细胞,可降低或消除患者中受HIV感染细胞,和/或可减少或消除先前和/或同时用其它已知抗逆转录病毒疗法治疗的患者中受HIV感染细胞的残余病灶。
其它实施方案涉及将T20小环用于基因疗法,如以下所详细讨论。其它实施方案涉及作为疫苗的组分的T20小环。在某些实施方案中,疫苗中T20或其它抗原的用途可通过使用佐剂来增强。在特定实施方案中,佐剂可为DDD部分(例如SEQ ID NO:5)。
附图简述
以下附图形成本说明书的一部分,并且被包括来进一步说明本发明的特定实施方案的某些方面。实施方案可通过参照这些附图中的一个或多个,结合本文呈现的详细描述来更充分了解。
图1公开体外由MC载体表达T20肽。对由用MC转染或未转染(对照)的HeLa细胞获得的免疫沉淀(IP)肽和细胞溶解产物进行蛋白质印迹。
图2显示介质、单独T20肽、单独DNA疫苗质粒gp160、或与质粒gp160混合的T20的鼻内(i.n.a)沉积的比较。与质粒混合的T20肽似乎使结合gp160的血清IgG效价以及针对HIV-1亚型B的中和抗体(NT)两者均增强。
图3显示不同T20核酸序列(SEQ ID NO:3),其并有DDD2部分(加下划线)、铰链接头(斜体)、(His)6GS(SEQ ID NO:16)(粗体)和T20(加下划线和斜体)。
图4显示在3次免疫之后,A组中的经免疫小鼠中抗T20抗体和抗gp140C抗体的产生。用T20和gp140C抗原,与来自A组小鼠的血清一起孵育来进行ELISA。结果指示质粒Env+小环T20成功产生gp140C特异性抗体与T20特异性抗体两者,并且在第3次免疫之后,效价增加约5倍。
图5显示在A组(质粒Env A、B和C+T20MC)中的经免疫小鼠相对于D组(质粒Env A、B和C+空pKCMV载体)中的经免疫小鼠之间的比较。ELISA按照图4的图例进行。
说明性实施方案的描述
本申请中引用的所有文件或文件的部分(包括但不限于专利、专利申请、文章、书籍和专著)都据此以引用的方式整体明确并入本文。
定义
如本文所用,“一(a/an)”可意指一个或超过一个条目。
如本文所用,术语“和”和“或”可用于意指联合(conjunctive)或分离(disjunctive)。也就是说,除非另外陈述,否则两个术语均应被理解为等效于“和/或”。
如本文所用,“约”意指在某一数目的正或负10%内。举例来说,“约100”将意指在90与110之间的任何数目。
如本文所用,“小环”是指通过诱导型重组酶来由质粒获得,但缺乏任何原核DNA的小(4kb或更少)环状双链附加型表达载体。小环可具有或可不具有例如通过并入S/MAR元件达成的自我复制性。
如本文所用,“脂多糖”是指包含由共价键接合的脂质和多糖的大分子。脂多糖通常见于革兰氏阴性细菌的外膜中。适用的脂多糖包括充当内毒素和/或在宿主中引发强烈免疫应答的那些。
“治疗剂”是适用于治疗疾病的原子、分子或化合物。治疗剂的实例包括抗体、抗体片段、药物、病毒抑制剂、毒素、酶、核酸酶、激素、免疫调节剂、反义寡核苷酸、小干扰RNA(siRNA)、螯合剂、硼化合物、光活性剂、染料和放射性同位素。其它适用的示例性治疗剂和方法公开于美国专利申请公布号20050002945、20040018557、20030148409和20050014207中,所述美国专利申请公布各自以引用的方式并入本文。
术语“pDNA”是指质粒DNA。
T20融合抑制剂肽
一些病毒,最特别是HIV,必须经受与宿主细胞膜的复杂融合过程以进入宿主细胞并繁殖(例如美国公布号20040049018)。在HIV的情况下,在繁殖期间,HIV病毒的外膜与CD4+T细胞的细胞膜融合(美国公布号20040049018)。T20是抗病毒融合抑制剂的类别中受到FDA核准供人使用的首个成员。由于施用T20,HIV的繁殖被阻断,并且不发生所引起的CD4+T细胞死亡(美国公布号20040049018)。
来自两个大型的在国际上进行的III期试验的数据指示相较于采用无T-20的组合疗法的那些患者,在24周时,与T-20的组合疗法在至少两倍百分比的患者中使血液中HIV降低至不可检测水平,并且提供改进免疫应答(美国公布号20040049018)。另外,历经24周,接受T-20的那些患者经历病毒学失败或复发的可能性较小(美国公布号20040049018)。
病毒对当前核准的抗HIV药物的抗性是当今临床HIV管理中的重大问题。用当前核准的药物开始组合抗逆转录病毒治疗的许多患者将随时间对这些药剂中的一种或多种产生抗性。然而,研究表明T20可不受对任何当前核准的抗逆转录病毒类别的抗性影响(美国公布号20040049018)。
由于恩夫韦地的肽特性,所以它以冻干形式销售,其必须由患者复原并每日两次通过皮下注射来自我施用。由于这种疗法的长期特性,所以对于患者对这个药物方案的依从性来说,这个剂型可为主要问题。本发明通过提供如下所述的小环形式的T20表达载体来避免对由患者进行每日皮下自我施用的需要。一旦T20MC已被转染或以其它方式引入宿主细胞中,小环设计的相对稳定性即使得长期暴露于T20肽,而无需重复和频繁施用。
本文公开的T20MC构建体可用于在培养的细胞中在体外产生T20肽,或可替代地被转染或以其它方式引入宿主细胞中,所述宿主细胞接着在以下更详细描述的基因治疗方法中在体内表达和分泌T20进入循环中。体外产生的T20可用于恩夫韦地的处于当前治疗使用中的相同方法和组合物中。
T20疫苗
已公开对基于树突细胞(DC)的疫苗的使用(参见例如2011年1月21日提交的美国公布号20110182937,附图和实施例章节以引用的方式并入本文)。树突细胞可通过标准技术来分离,并且通过转染或脂质体转染来用编码抗原的载体装载(美国公布号20110182937;Fong和Engleman,2000,Dendritic Cells in Cancer Immunotherapy.AnnRev Immunol)。克隆蛋白质或肽在DC中的表达会诱导对蛋白质或肽抗原的免疫应答。在某些实施方案中,可将T20MC载体装载至患者的经分离DC(或同种异体DC)中,并且向所述患者施用经装载DC(例如Kundu等,2009,AIDS Research and Human Retroviruses 14:551-60)。因为T20序列模拟在HIV中表达的天然gp41的C末端七联体重复2(HR2)区域的一部分,所以用T20MC转染的DC的施用有效诱导针对gp41和HIV的免疫应答,其适用于降低或消除病毒。
在替代性实施方案中,疫苗功效可例如通过使用辅助化合物诸如GM-CSF和/或干扰素-α2b来改进(美国公布号20110182937)。来自患者(或同种异体受试者)的血液样品可用于例如通过淘析来分离单核细胞。可接着在包含GM-CSF和/或干扰素-α2b的培养基中培养单核细胞,随后用T20MC装载活化的DC。在抗原装载和活化之后,可在约72小时收获DC疫苗,通过离心来洗涤并再混悬。在某些实施方案中,可将疫苗储存在由80%血浆电解质(PlasmaLyte)、10%人血清白蛋白(HSA)和10%DMSO组成的冷冻溶液中。在施用之前,可将细胞混悬液的等分试样填充至玻璃疫苗小瓶中并冷冻。
T20肽在DC中的表达有效诱导宿主免疫系统对HIV应答。在其它替代性实施方案中,DC可通过暴露于脂多糖来进一步活化(美国公布号20110182937)。疫苗疗法可单独或与抗逆转录病毒疗法诸如HAART、蛋白酶抑制剂、逆转录酶抑制剂或抗HIV抗体组合利用。
以可与给药制剂相容的方式,并且以诸如将为治疗有效和具有免疫原性的量施用疫苗。待施用的数量取决于待治疗的受试者,包括例如个体的免疫系统产生免疫应答的能力。细胞或活性成分的需要被施用的确切量取决于从业者的判断。然而,适于细胞疫苗的剂量范围是约数千个细胞至数百万个细胞的量级。对于标准表位或表位递送疫苗,每次疫苗接种,疫苗可包含数百微克或更少的抗原性肽。适于初始施用和加强注射的方案也是可变的,但典型的是初始施用继之以后续接种。尽管施用途径可变化,但递送途径可为口服、静脉内、皮下、腹膜或肌肉内。
在许多情况下,将合乎需要的是进行多次疫苗施用,例如4至6次疫苗接种,每周或每隔一周加以提供。正常疫苗接种方案将经常以2至12周间隔或3至6周间隔进行。在1-5年(通常3年)的间隔下的定期加强可合乎维持免疫应答的保护性水平的需要,或在可能缓解或再感染后合乎需要。
如下所讨论,在某些实施方案中,DDD肽可作为佐剂共同施用以增加对外来抗原诸如T20的免疫应答。DDD可单独施用,或更优选地,例如融合于T20以融合蛋白形式提供。
T20基因疗法
在基因疗法中,目的在于通过引入遗传物质,经常是编码蛋白质的DNA或治疗性RNA来改变细胞的行为。基因疗法已用于2142个临床试验中,其中癌症疾病是最常见靶标(Vectors used in Gene Therapy.Gene therapy Clinical Trials Worldwide 2015)。迄今为止存在两种可商购获得的核准的基因疗法药物-用于治疗头颈部鳞状细胞癌的含有肿瘤抑制基因的今又生(Gendicine)在2003年在中国被核准,并且靶向脂蛋白脂肪酶缺乏症的格利贝拉(Glybera)在2012年在欧洲被核准(参见例如Patil等,2005AAPS J 8:E61-77;Ferreira等,2014,Front Immunol 5:82)。
为改变细胞行为,遗传物质必须转运至细胞中并到达核。用以实现这个的两种主要方式是使用病毒载体,其中经工程改造的病毒携带治疗性DNA;或非病毒载体,其通常基于在细菌中产生的质粒。
病毒已特异性进化来将它们的基因组递送至靶细胞中;这是病毒如何传播的方式。通过改变由病毒转运的遗传物质的序列,以及改变病毒以防止它处于复制感受态,这个性状可被利用来达成基因疗法。病毒载体极其高效递送它们的运载物,因此已用于接近70%的基因疗法临床试验中(例如Edelstein等,2007,J Gene Med 9:833-42)。然而,使用病毒载体存在安全性问题(Edelstein等,2007,J Gene Med 9:833-42)。因为它们源于人病原体,所以它们可引发免疫应答,这是因为患者已遭遇天然病毒株,因此携带针对载体的抗体,或是因为维持治疗性基因的表达需要重复治疗。这是腺病毒和AAV源性载体的情况,其中转基因以附加体形式存在于细胞中,并且将随着细胞分裂而稀释(Edelstein等,2007,JGene Med 9:833-42)。
其它病毒诸如逆转录病毒将它们的基因组整合至靶细胞的基因组中,并且这些病毒可用治疗性序列工程改造来进行相同举动。这使得能够在细胞和所有子代细胞中进行恒定表达,而无需再施用载体。然而,整合事件可导致可具有不利影响的突变(Edelstein等,2007,J Gene Med 9:833-42)。在使用逆转录病毒载体治疗X染色体连锁的重度联合免疫缺陷时,整合导致致癌基因被活化,并且临床试验中总计20名患者中的5名罹患白血病(Hacein-Bey-Abina,2003,Science 302:415-19;Hacein-Bey-Abina,2008,J Clin Invest118:3132-42;Howe等2008,J Clin Invest 118:3143-50)。病毒载体的另一限制是尺寸;对病毒衣壳中可被转运的遗传物质的量存在物理限制。
相比于病毒载体,非病毒载体通常被视为更安全以及更易于产生,但在递送和长期表达方面的效率较低。这被认为部分地归因于质粒骨架,即仅为在细菌中增殖所需的序列诸如复制起点和选择标记(通常是抗生素抗性基因)(Vandermeulen等,2011,Mol Ther19:1942-49)。相比于真核DNA,细菌产生的DNA序列具有不同甲基化样式。已显示这可诱导免疫应答,尤其在与用脂质转染进行组合的情况下(参见例如Bessis等,2004,Gene Ther11:S10-17)。此外,对于裸露递送质粒,因为裸露DNA缺乏主动转运系统来递送至细胞中以及转移至核中,所以非病毒载体远不及病毒载体高效,并且表达是低下和瞬时的,此可部分地归因于表观遗传现象。
存在用以改进这些载体的效率的许多不同方法。可将质粒与脂质和聚合物包装在一起,此使DNA紧缩,并且保护它免遭降解,以及有助于通过与细胞膜的脂质双层融合来摄取(例如Dincer等,2005,Gene Therapy 12:S139-45)。用以使跨越细胞膜和核膜的转运增加的另一化学方法在于使质粒构建体与细胞渗透肽或核配体序列连接(例如Jarver和Langel,2004,Drug Discovery Today 9:395-402)。物理递送方法使用不同推动力来增强向靶细胞中的转运,诸如电穿孔、气动或高体积输注(Kamimura等,2011,Pharm Med 25:293-306)。
一种用以优化质粒载体的方式在于通过在产生细菌中重组来移除细菌骨架。所得载体被称为小环(MC)(参见例如Nehlsen等,2006,Gene Ther Mol Biol 10:233-44)。相较于质粒或病毒载体,MC的较小尺寸使得能够采用较高剂量,延长表达和增加稳健性。MC构建体缺乏细菌序列和抗生素抗性基因的事实使得MC载体成为非病毒基因疗法的具有吸引力的替代方案。在以下工作实施例中,提供表达T20的MC构建体。
T20小环载体
本发明的各种实施方案涉及呈小环构建体形式的T20表达载体。已公开若干产生小环的系统。首个系统基于用于亲本质粒的重组的λ噬菌体整合酶(Darquet等,1997,GeneTher 4:1341-49)。随后的系统由Bigger等(2001,J Biol Chem 276:23018-27)开发,并且利用具有侧接于表达盒的LoxP位点的Cre重组酶表达系统。然而,在重组之后loxP位点仍然具有活性,且小环可由于盒再次反向重组成亲本质粒而丢失,并且必须使位点突变以确保单向性。Mayrhofer等(2008,J Gene Med 10:1253-69)公开一种基于ParA解离酶的系统,在MC构建体中添加细菌乳糖操纵子位点,并且使用亲和色谱法来纯化载体。在所有这些系统中,在重组之后,亲本质粒和小质粒都保持在细菌中,并且因为它们含有复制起点,所以有可能它们将继续在细菌中扩增,从而将小环稀释。
以下实施例中使用的系统由Chen等(2003,Mol Ther 8:495-500;2005,Hum GeneTher 16:126-31)开发,其中链霉菌属(Streptomyces)模板整合酶ΦC3用于重组,并且表达盒由attB和attP重组位点侧接。分别在小环和小质粒中的所得attR和attL位点不能再次重组来重新形成亲本质粒。使MC与非所需产物-小质粒和任何未重组亲本质粒分离的困难通过在质粒骨架中在重组位点外部引入罕见限制酶识别位点,以及提供所述限制酶的基因来解决。在以下公开的初始研究中,我们使用这个系统的较早形式,其中整合酶和限制酶的基因由亲本质粒编码。两种基因均被置于阿拉伯糖诱导型启动子的控制下。这个方法产生巨大亲本质粒,但优势是可使用任何重组感受态大肠杆菌(Escherichia coli/E.coli)产生菌株。然而,大多数细菌具有全部阿拉伯糖输入作用或不具有阿拉伯糖输入作用,其中在诱导主动输入之前在细菌内部的阿拉伯糖的水平需要达到阈值水平(Siegele等,1997,ProcNatl Acad Sci USA 94:8168-72)。这产生从未达到这个阈值的细菌子群体,其中重组和消化均未被诱导,从而导致所需小环产物受非所需质粒污染。亲本质粒和小质粒含有复制起点,因此逃脱降解的任何拷贝都可继续复制。
ΦC31系统随后通过将来自亲本质粒的基因移动至具有组成型活性的阿拉伯糖转运的细菌菌株的细菌基因组中来精制(Kay等,2010,Nat Biotechnol 28:1287-89)。这产生更小和更稳定的亲本质粒以及更纯的小环部分。这个系统用于以下显示的随后研究中。
已显示电穿孔的效率与质粒DNA构建体的尺寸相关(Molnar等,2004,Mol Ther10:447-55),并且较低尺寸对于体外(Kreiss等,1999,Nucleic Acids Res 27:3792-98)和体内(Loisel等,2001,J Liposome Res 11:127-138)脂质体转染也有益。Kreiss等(1999)讨论pDNA的尺寸可影响DNA从脂质复合物释放的机理或DNA在细胞内穿过细胞质向核中的迁移,或影响这两者。McLenachan等(2007,Genomics 89:708-20)研究将在5至200千碱基对(kbp)的范围内的质粒DNA递送至小鼠胚胎干细胞中,并且报道当使用较小构建体时,核递送的尺寸依赖性增加。
Fogg等(2006,J Phys Condens Matter 18:S145-59)研究在250至1000bp的范围内的MC的重组。在较小尺寸下,他们报道MC倾向于形成多联(concatameric)构建体而非单体,并且提出这归因于分子间而非分子内重组事件。他们的在序列长度与分子内重组效率之间存在明确反向关系的观察结果与我们的观察结果一致。
某些实施方案涉及通过向患者施用T20MC以使得体内产生和分泌T20肽来进行的基因疗法。使得身体产生基于它自身蛋白质的药物是一种有吸引力的想法。Yi等(2014,SciRep 4:5961)已使用具有用于类风湿性关节炎的两种基于蛋白质的药物依那西普(etanercept)和托珠单抗(tocilizumab)的核苷酸序列的MC探究这个可能性。他们报道在关节炎小鼠模型中静脉内注射MC之后,这些自身产生的药物具有功能活性。另一研究在糖尿病动物模型中评价MC。Alam等(2013,PLoS One 8:e67515)使用MC构建体来进行受葡萄糖调节的胰岛素表达,所述胰岛素被递送至糖尿病大鼠的肝中。他们报道归一化的重量增加,并且治疗使各种糖尿病相关的代谢调节异常标志物恢复。MC构建体也由Park等(2006,JControl Release 114:118-25)通过递送脂联素(adiponectin)的基因来用于治疗肥胖小鼠的胰岛素抗性。
MC形式已被口服递送,配制成壳聚糖纳米粒子,以诱导经修饰的因子IX在乙型血友病小鼠中的小肠中表达(Quade-Lyssy等,2014,JTH 12:932-42)。瞬时局部转基因表达、临床相关因子IX活性水平和部分表型纠正通过口服基因疗法得以实现,在重复施用后没有不利免疫应答的迹象(Quade-Lyssy等,2014,JTH 12:932-42)。
Hyun等(20l3,Stem Cells Transl Med 2:690-702)通过表达调节线粒体凋亡路径的促存活蛋白Bcl-2来使用MC载体促进干细胞存活。当用MC处理干细胞,接着将经转染细胞递送至创伤中时,所述促存活蛋白得以过表达,并且骨形成得以增强(Hyun等2013,StemCells Transl Med 2:690-702)。也有研究显示MC适于转染干细胞,因此对它们进行转基因修饰,同时保留它们随后分化的能力,从而表明当瞬时表达足以刺激组织再生时,MC可能是适于干细胞疗法的载体(Madeira等,2013,Biomacromolecules 14:1379-87)。
其中MC载体已被频繁使用的领域是癌症研究。肿瘤具有增强的可渗透性和保留作用,此使得达成进入与在肿瘤内部隔离诸如脂质的大分子两者。Chang等(2014,BioMed ResIntl 2014:156356)利用这个性质来将脂质复合MC载体递送至小鼠中乙型肝炎病毒诱发的肝细胞癌瘤中。MC编码转移抑制性雄激素受体,并且可持续多达60天检测到转基因蛋白(Chang等,2014,BioMed Res Intl 2014:156356)。Wu等(2006,Clin Cancer Res 12:4702-13)研究在体外以及在体内在小鼠中的鼻咽癌异种移植物中由MC载体表达肿瘤坏死因子α,并且显示体外表达良好以及体内异种移植物肿瘤生长降低和存活延长,但当使用相同重量剂量时,MC仅在体内优于大型亲本质粒(Wu等,2006,Clin Cancer Res 12:4702-13)。他们显示在治疗之后持续21天由MC在肿瘤中进行表达,随时间衰退,而由大型亲本质粒进行的表达在7天之后几乎丧失(Wu等,2006,Clin Cancer Res 12:4702-13)。同一研究组随后构建编码血管生成抑制剂内皮抑素的MC,并且在相同鼻咽癌异种移植物模型中对它进行评价(Xu等,2012,Cancer Gene Ther 19:110-17)。他们报道持续实验的完整20天肿瘤生长降低以及在肿瘤内注射之后肿瘤血管化降低(Xu等,2012,Cancer Gene Ther 19:110-17)。
在一项引起关注的癌症疗法研究中,Gaspar等(2014,J Control Release 189:90-104)使用胶束纳米载体来共同递送编码肿瘤坏死因子α相关的凋亡诱导性配体的MC和化学治疗药物多柔比星(doxorubicin)。他们观察到良好体外摄取和在相对低浓度下小鼠中的抗肿瘤作用(Gaspar等,2014,J Control Release 189:90-104)。
编码shRNA的miMC已用于肌肉中,并且也用作防止病毒复制的抑制剂的表达载体。在一项由Yang等(2012,Antiviral Res 96:234-44)进行的研究中,两种作为手足口病的主要病因的病毒被靶向。由miMC载体对shRNA的表达在体外以及在受病毒感染的小鼠模型中阻断这些病毒的复制和基因表达,伴有受感染小鼠的症状缓和(Yang等,2012,AntiviralRes 96:234-44)。在这个研究中,Yang等通过在不同方向上用两个不同启动子侧接序列来设计一种用于由相同编码序列表达两个shRNA的系统,因为snRNA在有义和反义上是对称的。
蛋白质表达载体的一特殊情况是设计用于DNA疫苗接种的质粒。属于使用质粒的优势的是相较于常规疫苗制造,开发与产生两者均简易。此外,已知DNA疫苗在室温下极其稳定,此对于运输与储存两者均具有重要性(Quaak等,2010,AAPS PharmSciTech 11:344-50)。因为抗原由pDNA在靶细胞内表达,所以所得肽更可能类似于例如病毒蛋白质的具有所有必要翻译后修饰的天然形式。
在一则由Dietz等(2013,Mol Ther 21:1526-35)进行的报道中,MC构建体显示作为DNA疫苗载体的前景。MC在体外以及在体内在小鼠中具有更高和更延长表达,以及在体内具有增强的免疫原性。在一激发实验中,在李氏杆菌病(listeriosis)小鼠模型中,MC载体赋予更好保护,并且引发更强烈抗原特异性CD8+T细胞应答(Dietz等,2013,Mol Ther 21:1526-35)。CD8+T细胞是针对任何病毒性感染的细胞免疫应答的重要组分,因为它们可识别并消除受感染细胞。因此,强烈CD8+T细胞应答对于HIV疫苗接种可具有重要性。HIV主要靶向CD4+T细胞,并且通过由此影响这些细胞的功能,HIV损害CD8+T细胞的成熟(Gulzar和Copeland,2004,Current HIV Res 2:23-37)。
Wang等(2014,J Virol 88:1924-34)出于疫苗接种目的使用表达HIV蛋白的MC。在他们的实验中,当使用MC时,体液和细胞免疫应答是常规质粒的应答的两倍。值得注意的是,在他们的研究中,他们也观察到相较于单独肌肉内注射、与或不与电穿孔一起进行真皮内注射、或高体积肝灌注,与体内电穿孔一起进行肌肉内MC注射诱导最强烈体液和细胞免疫应答(Wang等,2014,J Virol 88:1924-34)。Wang等也报道当使用电穿孔时,MC载体的表达改进比常规质粒的表达改进高4倍。
使用治疗性小环的这些和其它已知方法和组合物可用于实施要求保护的本发明。
组合疗法
如上所讨论,用T20肽和/或小环进行的疗法可通过与其它抗病毒剂组合来增强。众多所述药剂在本领域中是已知的,包括但不限于阿巴卡韦、氨多索韦(amdoxovir)、阿立他滨(apricitabine)、阿扎那韦、贝韦立马(bevirimat)、卡拉诺利A(calanolide A)、CCR5、CD4、塞拉集宁(ceragenin)、可比司他(cobicistat)、蓝藻抗病毒蛋白-N(cyanovirin-N)、地瑞那韦、二芳基嘧啶(diarylpyrimidine)、地达诺新、度鲁特韦、依法韦仑、埃替格韦、艾夫他滨(elvucitabine)、恩曲他滨、没食子酸表没食子儿茶素(epigallotachen gallate)、费替那韦(festinavir)、福沙那韦(fosamprenavir)、膦甲酸(foscamet)、格瑞弗森(griffithsin)、格鲁博南A(globoidnan A)、羟基脲(hydroxycarbamide)、茚地那韦、KP-146、拉米夫定、来非那韦(lefinavir)、来司韦林(lersivirine)、洛匹那韦、米替福新(miltefosine)、MK-2048、奈非那韦、奈韦拉平、拉西韦(racivir)、雷特格韦、利托那韦、沙奎那韦(saquinavir)、塞利西利(selicicib)、司他福定(stafudine)、司他匹定(stampidine)、司他夫定、Tat拮抗剂、替诺福韦、替拉那韦(tipranavir)、天花粉蛋白(trichosanthin)、TRIM5α、维康(vivecon)、扎西他滨、齐多夫定或齐多夫定。
一种类型的抗HIV剂包括能够破坏或抑制HIV的感染性和/或病毒性的中和抗体或其片段。多种HIV中和抗体在本领域中是已知的,并且可使用任何所述已知抗体或其片段,包括但不限于P4/D10、2G12(例如Joos等,Antimicrob Agents Chemother 2006,50:1773-79)、4E10(Joos等,2006)、2F5(Joos等,2006)、b12(例如Wu等,J Virol 2006,80:2585)、X5(Moulard等,Proc Natl Acad Sci 2002,99:6913-18)或其任何组合。当使用多特异性抗体或片段或单特异性抗体的组合时,熟练技术人员将认识到可使结合相同或不同HIV表位的多种抗体或片段组合。尽管针对HIV包膜蛋白(gp120)和/或gp41的抗体是优选的,但熟练技术人员将认识到其它HIV靶抗原可用于产生将靶向受HIV感染细胞的抗体或其片段。在一些情况下,可利用结合一种或多种HIV抗原与T细胞抗原(例如CD4、CCR5和/或CXCR4)组合的抗体或片段。
另一类别的潜在治疗剂由聚集体抑制剂组成。聚集体是被认为应答于错误折叠蛋白质而形成的大型细胞内复合物(参见例如Heath等,J.Cell Biol.153:449-55,2001;Johnstone等,J.Cell Biol.143:1883-98,1998;Wileman,Science 312:875-78,2006)。最近,已表明聚集体可在病毒粒子装配方面起作用(Heath等,2001;Wileman,2006)。因此,聚集体抑制剂可起阻断或抑制从受HIV或其它病毒感染的细胞形成新感染性病毒粒子的作用。多种聚集体抑制剂是已知的,诸如ALLN、诺考达唑(nocodazole)、秋水仙碱(colchicine)和长春花碱(vinblastine)(Johnston等,1998)、其它微管抑制剂(Gerdes和Katsanis,Hum.Molec.Genet.14:R291-300,2005);硼替佐米(bortezomib)(Catley等,Blood108:3441-49,2006)、图巴新(tubacin)、组蛋白脱乙酰基酶抑制剂(Corcoran等,Curr.Biol.14:488-92,2004),并且可使用任何所述已知聚集体抑制剂。
在各种实施方案中,可使用一种或多种免疫调节剂。如本文所用,术语“免疫调节剂”包括细胞因子、干细胞生长因子、淋巴细胞毒素和造血因子,诸如白介素、集落刺激因子、干扰素(例如干扰素-α、干扰素-β和干扰素-γ)和指定为“S1因子”的干细胞生长因子。合适的免疫调节剂部分的实例包括IL-2、IL-6、IL-10、IL-12、IL-18、IL-21、干扰素-γ、TNF-α等。
术语“细胞因子”是由一个细胞群体释放的作为细胞间介体作用于另一细胞的蛋白质或肽的通用术语。如本文广泛所用,细胞因子的实例包括淋巴因子、单核因子、生长因子和传统多肽激素。包括在细胞因子之中的是生长激素,诸如人生长激素、N-甲硫氨酰基人生长激素和牛生长激素;甲状旁腺激素;甲状腺素(thyroxine);胰岛素;胰岛素原;松弛素(relaxin);松弛素原(prorelaxin);糖蛋白激素,诸如卵泡刺激激素(FSH)、甲状腺刺激激素(TSH)和促黄体生成激素(luteinizing hormone,LH);肝生长因子;前列腺素、纤维母细胞生长因子;促乳素;胎盘催乳素、OB蛋白;肿瘤坏死因子-α和肿瘤坏死因子-β;缪勒抑制物质(mullerian-inhibiting substance);小鼠促性腺激素相关肽;抑制素(inhibin);活化素(activin);血管内皮生长因子;整合素;血小板生成素(TPO);神经生长因子,诸如NGF-β;血小板生长因子;转化生长因子(TGF),诸如TGF-α和TGF-β;胰岛素样生长因子-I和胰岛素样生长因子-II;红细胞生成素(EPO);骨诱导性因子;干扰素,诸如干扰素-α、干扰素-β和干扰素-γ;集落刺激因子(CSF),诸如巨噬细胞-CSF(M-CSF);粒细胞-巨噬细胞-CSF(GM-CSF);和粒细胞-CSF(G-CSF);白介素(IL)(诸如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12;IL-13、IL-14、IL-15、IL-16、IL-17、IL-18、IL-21)、LIF、G-CSF、GM-CSF、M-CSF、EPO、kit配体或FLT-3、血管抑素、血栓反应素(thrombospondin)、内皮抑素、肿瘤坏死因子和LT。如本文所用,术语细胞因子包括来自天然来源或来自重组细胞培养的蛋白质以及天然序列细胞因子的生物活性等效物。
趋化因子通常作为化学引诱剂起使免疫效应细胞募集至趋化因子表达部位的作用。可有利的是表达特定趋化因子基因与例如细胞因子基因组合以增强使其它免疫系统组分募集至治疗部位。趋化因子包括但不限于RANTES、MCAF、MIP1-α、MIP1-β和IP-10。熟练技术人员将认识到某些细胞因子也已知具有化学引诱作用,并且也可被分类在术语趋化因子下。类似地,术语免疫调节剂和细胞因子在它们的各自成员方面具有重叠。
T20融合抑制剂可潜在与不同融合抑制剂组合使用。HIV融合抑制剂描述于PCT专利申请公布号WO 2007045463中。已知由于天然存在的多态性,所以在不同HIV病毒株之间gp41蛋白的氨基酸序列不同。但可认定相同结构域构造,即融合信号、两个七联体重复结构域(HR1、HR2)和跨膜结构域。融合(或促融)结构域参与对细胞膜的插入和崩解。具有从gp41的HR1或HR2结构域推导的氨基酸序列的肽是HIV摄取至细胞中的有效体外和体内抑制剂(参见例如美国专利号5,464,933;5,656,480;6,258,782;6,348,568;6,656,906)。举例来说,T20、HR2肽和T651(美国专利号6,479,055)是HIV感染的强力抑制剂。已试图增强HR2源性肽的功效,例如通过氨基酸取代或化学交联(Sia等,2002,PNAS USA 99:14664-14669;Otaka等,2002,Angew.Chem.Int.41:2937-2940)。
示例性抗促融肽见于美国专利号5,464,933;5,656,480;6,013,263;6,017,536;6,020,459;6,093,794;6,060,065;6,258,782;6,348,568;6,479,055;6,656,906;以及PCT专利申请公布号WO 1996/19495、WO 1996/40191、WO 1999/59615、WO 2000/69902和WO2005/067960中,所述专利各自的实施例章节以引用的方式并入本文。
在某些实施方案中,小环或其它载体可用于递送siRNA或干扰RNA物质。已报道siRNA的多种载体部分,并且可使用任何所述已知载体。载体的非限制性实例包括鱼精蛋白(Rossi,2005,Nat Biotech 23:682-84;Song等,2005,Nat Biotech 23:709-17);树枝状聚合物诸如PAMAM树枝状聚合物(Pan等,2007,Cancer Res.67:8156-8163);聚乙烯亚胺(Schiffelers等,2004,Nucl Acids Res 32:e149);聚丙稀亚胺(Taratula等,2009,JControl Release 140:284-93);聚赖氨酸(Inoue等,2008,J Control Release 126:59-66);含有组氨酸的可还原聚阳离子(Stevenson等,2008,J Control Release 130:46-56);组蛋白H1蛋白(Haberland等,2009,Mol Biol Rep 26:1083-93);阳离子梳型共聚物(Sato等,2007,J Control Release 122:209-16);聚合胶束(美国专利申请公布号20100121043);和壳聚糖-焦磷酸硫胺素(Rojanarata等,2008,Pharm Res 25:2807-14)。熟练技术人员将认识到一般来说,聚阳离子蛋白质或聚合物适用作siRNA载体。熟练技术人员将进一步认识到siRNA载体也可用于携带其它寡核苷酸或核酸物质诸如反义寡核苷酸或短DNA基因。
许多siRNA物质可从已知来源商购获得,诸如Sigma-Aldrich(St Louis,MO)、Invitrogen(Carlsbad,CA)、Santa Cruz Biotechnology(Santa Cruz,CA)、Ambion(Austin,TX)、Dharmacon(Thermo Scientific,Lafayette,CO)、Promega(Madison,WI)、Mirus Bio(Madison,WI)和Qiagen(Valencia,CA)以及许多其它来源。siRNA物质的其它可公开获得的来源包括在Stockholm Bioinformatics Centre的siRNAdb数据库、MIT/ICBPsiRNA数据库、在Broad Institute的RNAi Consortium shRNA文库和在NCBI的Probe数据库。举例来说,在NCBI Probe数据库中存在30,852个siRNA物质。熟练技术人员将认识到对于任何目标基因,siRNA物质已被设计,或可易于使用可公开获得的软件工具加以设计。
在某些实施方案中,抗原性融合蛋白或复合物可通过DOCK-AND-技术来形成(参见例如美国专利号7,521,056;7,527,787;7,534,866;7,550,143和7,666,400,所述专利各自的实施例章节以引用的方式并入本文)。通常,技术利用发生在cAMP依赖性蛋白激酶(PKA)的调节(R)亚基的二聚化和对接结构域(DDD)序列与源于多种AKAP蛋白中的任一种的锚定结构域(AD)序列之间的特异性和高亲和力结合相互作用(Baillie等,FEBSLetters.2005;579:3264.Wong和Scott,Nat.Rev.Mol.Cell Biol.2004;5:959)。DDD和AD肽可连接于任何蛋白质、肽或其它分子。因为DDD序列自发二聚化并结合AD序列,所以技术使得在可连接于DDD或AD序列的任何所选分子之间形成复合物。尽管标准复合物包含两个DDD连接的分子连接于一个AD连接的分子的三聚体,但在复合物结构方面的变化使得形成二聚体、三聚体、四聚体、五聚体、六聚体和其它多聚体。复合物或融合蛋白可包含一种或多种其它效应物,诸如蛋白质、肽、抗体、抗体片段、免疫调节剂、细胞因子、白介素、干扰素、结合蛋白、肽配体、载体蛋白、毒素、核糖核酸酶诸如豹蛙酶、抑制性寡核苷酸诸如siRNA、抗原或异种抗原、聚合物诸如PEG、酶、治疗剂、激素、细胞毒性剂、抗血管生成剂、促凋亡剂或任何其它分子或聚集物。
在由第二信使cAMP结合R亚基触发的一个最充分研究的信号转导路径中起主要作用的PKA首先在1968年从兔骨骼肌分离(Walsh等,J.Biol.Chem.1968;243:3763)。全酶的结构由两个以非活性形式由R亚基固持的催化亚基组成(Taylor,J.Biol.Chem.1989;264:8443)。发现PKA的同功酶具有两种类型的R亚基(RI和RII),并且各类型具有α和β亚型(Scott,Pharmacol.Ther.1991;50:123)。因此,PKA调节亚基的四个亚型是RIα、RIβ、RIIα和RIIβ。R亚基已仅以稳定二聚体形式被分离,并且已显示二聚化结构域由RIIα的前44个氨基末端残基组成(Newlon等,Nat.Struct.Biol.1999;6:222)。如下所讨论,其它调节亚基的氨基酸序列的类似部分参与二聚化和对接中,各自位于调节亚基的N末端附近。cAMP结合R亚基导致释放用于达成广谱丝氨酸/苏氨酸激酶活性的活性催化亚基,其通过PKA与AKAP对接达成的PKA区室化来朝向所选底物进行定向(Scott等,J.Biol.Chem.1990;265;21561)。
自从在1984年表征首个AKAP,即微管相关蛋白-2(Lohmann等,Proc.Natl.Acad.Sci USA.1984;81:6723)以来,已在酵母至人的范围内的物种中鉴定超过50种定位于各种亚细胞部位(包括质膜、肌动蛋白细胞骨架、核、线粒体和内质网),具有不同结构的AKAP(Wong和Scott,Nat.Rev.Mol.Cell Biol.2004;5:959)。AKAP的针对PKA的AD是具有14-18个残基的两亲性螺旋(Carr等,J.Biol.Chem.1991;266:14188)。AD的氨基酸序列在个别AKAP之间有变化,其中对RII二聚体报道的结合亲和力在2至90nM的范围内(Alto等,Proc.Natl.Acad.Sci.USA.2003;100:4445)。AKAP将仅结合二聚R亚基。对于人RIIα,AD结合由23个氨基末端残基形成的疏水性表面(Colledge和Scott,Trends Cell Biol.1999;6:216)。因此,人RIIα的二聚化结构域与AKAP结合结构域两者均位于在本文中称为DDD的相同N末端44个氨基酸的序列内(Newlon等,Nat.Struct.Biol.1999;6:222;Newlon等,EMBOJ.2001;20:1651)。
我们已开发用以利用人PKA调节亚基的DDD和AKAP的AD作为一对卓越接头模块来将下文称为A和B的任何两个实体对接成非共价复合物的平台技术,所述非共价复合物可通过将半胱氨酸残基引入DDD与AD两者中的策略位置处以有助于形成二硫键来进一步锁定成复合物。所述方法的一般性方法学如下。通过使DDD序列连接于A的前体,从而产生下文称为a的第一组分来构建实体A。因为DDD序列将实现自发形成二聚体,所以A将因此由a2组成。通过使AD序列连接于B的前体,从而产生下文称为b的第二组分来构建实体B。a2中含有的DDD的二聚基序将产生用于结合b中含有的AD序列的对接位点,因此有助于a2和b即时缔合以形成由a2b组成的二元三聚复合物。以用以通过二硫桥来共价固定两个实体的随后反应使得这个结合事件不可逆,因为初始结合相互作用将使放置在DDD与AD两者上的反应性硫醇基团邻近(Chmura等,Proc.Natl.Acad.Sci.USA.2001;98:8480)以进行位点特异性连接,所以基于有效局部浓度的原理,所述反应极其高效发生。使用接头、衔接头模块和前体的各种组合,可产生并使用广泛多种具有不同化学计量的构建体(参见例如U.S.No.7,550,143;7,521,056;7,534,866;7,527,787和7,666,400)。
通过远离两个前体的官能团来连接DDD和AD,也预期所述位点特异性连接会保持两个前体的原始活性。这个方法在性质上是模块化的,并且潜在地可应用于位点特异性和共价连接广泛范围的具有广泛范围的活性的物质,包括肽、蛋白质、抗体、抗体片段和其它效应物部分。利用以下实施例中描述的构建AD和DDD缀合效应物的融合蛋白方法,可将实际上任何蛋白质或肽并入构建体中。然而,技术不是限制性的,并且可利用其它缀合方法。
已知多种用于制备融合蛋白的方法,包括核酸合成、杂交和/或扩增以产生编码目标融合蛋白的合成双链核酸。可通过标准分子生物学技术将所述双链核酸插入表达载体中以产生融合蛋白(参见例如Sambrook等,Molecular Cloning,A laboratory manual,第2版,1989)。在所述优选实施方案中,AD和/或DDD部分可连接于效应物蛋白质或肽的N末端或C末端。然而,熟练技术人员将认识到视效应物部分和效应物部分的涉及于它的生理活性中的部分的化学性质而定,AD或DDD部分连接于所述效应物部分的位点可变化。可使用本领域中已知的技术,诸如使用二价交联试剂和/或其它化学缀合技术来进行多种效应物部分的位点特异性连接。
在某些实施方案中,在以下实施例中更详细讨论,可将DDD部分与另一抗原性肽或蛋白质一起并入融合蛋白中以诱导针对靶抗原的更强烈免疫应答。在特定实施方案中,示例性融合蛋白包含通过接头连接于T20肽的DDD2部分。因为T20序列模拟HIV的gp41蛋白的一部分,所以将融合蛋白或可产生融合蛋白的表达载体引入宿主中可诱导可防止HIV感染的免疫应答,或可诱导可抑制现有HIV感染的免疫应答。如下所讨论,其它DDD部分是已知的,并且可用于构建抗原性融合蛋白。普通技术人员将认识到与DDD部分组合使用以诱导免疫应答的靶抗原不限于T20。也可将本领域中已知的针对疾病(诸如癌症、自体免疫疾病或病原体感染)疗法的其它靶抗原连同DDD部分一起并入主题融合蛋白中。
AD和DDD部分中的结构-功能关系
DDD1
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:4)
DDD2
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO:5)
AD1
QIEYLAKQIVDNAIQQA(SEQ ID NO:6)
AD2
CGQIEYLAKQIVDNAIQQAGC(SEQ ID NO:7)
熟练技术人员将认识到DDD1和DDD2是基于蛋白激酶A的人RIIα亚型的DDD序列。然而,在替代性实施方案中,DDD和AD部分可基于蛋白激酶A的人RIα形式的DDD序列和相应AKAP序列,如在以下以DDD3、DDD3C和AD3所例示。
DDD3
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ ID NO:8)
DDD3C
MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ ID NO:9)
AD3
CGFEELAWKIAKMIWSDVFQQGC(SEQ ID NO:10)
在其它替代性实施方案中,AD和/或DDD部分的其它序列变体可用于构建复合物。举例来说,仅存在人PKA DDD序列的四个变体,对应于PKARIα、RIIα、RIβ和RIIβ的DDD部分。RIIαDDD序列是以上公开的DDD1和DDD2的基础。以下显示四个人PKA DDD序列。DDD序列代表以下残基:RIIα的1-44、RIIβ的1-44、RIα的12-61和RIβ的13-66。(应注意DDD1的序列从人PKARIIαDDD部分加以略微修饰。)任何所公开DDD序列或其它已知DDD序列都可用于构建具有增强的抗原性活性的融合蛋白或替代性构建体。
PKA RIα
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK(SEQ ID NO:11)
PKA RIβ
SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA(SEQ ID NO:12)
PKA RIIα
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ(SEQ ID NO:13)
PKA RIIβ
SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER(SEQ ID NO:14)
配制和施用
可进一步配制本文公开的抗HIV治疗剂以获得包括一种或多种药学上适合的赋形剂、一种或多种额外成分或这些的某一组合的组合物。这些可通过用以制备药学上适用的剂量的已知方法来实现,借此将活性成分与一种或多种药学上适合的赋形剂组合成混合物。无菌磷酸盐缓冲盐水是药学上适合的赋形剂的一个实例。其它适合赋形剂为本领域人员所熟知。参见例如Ansel等,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS,第5版(Lea和Febiger 1990);以及Gennaro(编),REMINGTON’S PHARMACEUTICALSCIENCES,第18版(Mack Publishing Company 1990)及其修订版。
一种用于施用本文所述的组合物的途径是胃肠外注射。在胃肠外施用中,组合物将与药学上可接受的赋形剂联合被配制成单位剂量可注射形式,诸如溶液剂、混悬剂或乳剂。所述赋形剂是固有无毒以及非治疗性的。所述赋形剂的实例是盐水、林格氏溶液(Ringer′s solution)、右旋糖溶液和汉克氏溶液(Hank′s solution)。也可使用非水性赋形剂,诸如不挥发性油和油酸乙酯。赋形剂可含有少量添加剂,诸如增强等张性和化学稳定性的物质,包括缓冲剂和防腐剂。
经配制组合物可通过例如弹丸注射或连续输注来用于静脉内施用。其它施用方法包括皮下、肌肉内、血管内或局部输注或通过口服递送。供施用的组合物可以单位剂型呈现,例如于安瓿中或在添加防腐剂的情况下于多次剂量容器中。组合物也可采用诸如于油性或水性媒介物中的混悬剂、溶液剂或乳剂的形式,并且可含有配制剂,诸如助悬剂、稳定剂和/或分散剂。或者,组合物可呈用于在使用之前用合适的媒介物例如无菌无热原水复原的粉末形式。
组合物可以溶液形式施用。其制剂应呈具有合适的药学上可接受的缓冲剂诸如磷酸盐、三(羟甲基)氨基甲烷盐酸盐或柠檬酸盐等的溶液形式。缓冲剂浓度应在1至100mM的范围内。经配制溶液也可以50至150mM的浓度含有盐诸如氯化钠或氯化钾。也可包括有效量的稳定剂,诸如甘油、白蛋白、球蛋白、清洁剂、明胶、鱼精蛋白或鱼精蛋白的盐。
试剂盒
一些实施方案涉及用于实施所要求保护的方法的试剂盒。试剂盒可包括T20小环或所表达的肽。可将试剂盒组分包装至容器中,诸如含有组合物的适于复原的无菌冻干制剂的小瓶。试剂盒也可含有一种或多种适于复原和/或稀释其它试剂的缓冲剂。可使用的其它容器包括但不限于袋、盘、盒、管等。试剂盒组分可被无菌包装和维持在容器内。可包括的另一组分是供使用试剂盒来达成它的用途的人士使用的说明书。
实施例
包括以下实施例以说明本发明的优选实施方案。本领域技术人员应了解随后实施例中公开的技术代表被发现在实施本发明方面良好地起作用的技术,因此可被视为构成它的优选实施模式。然而,本领域技术人员鉴于本公开应了解可在不脱离本发明的精神和范围下,在所公开的特定实施方案中进行许多改变,并且仍然会获得相似或类似结果。
实施例1.与T20MC相关的一般性方法和组合物
MC载体产生-最初,将含有CMV启动子、hVEGF-165的基因和兔β-球蛋白多聚腺苷酸化位点的表达盒从phVEGF 165载体克隆至p2ΦC31MC产生质粒中的两个重组位点之间。这是由Chen等(2003,Mol Ther 8:495-500;2005,Hum Gene Ther 16:126-31)开发的第一代ΦC3MC产生系统。在这个系统中,重组酶和核酸内切酶的诱导性基因位于亲本质粒上,并且可在任何重组感受态大肠杆菌产生菌株中进行产生。在过夜增殖经转导的细菌之后,添加阿拉伯糖诱导控制链霉菌噬菌体ΦC31整合酶基因和I-Sec-I核酸内切酶基因的pBAD启动子。因此,诱导亲本质粒重组成MC和小质粒,以及小质粒在位于重组位点外部的识别位点处由核酸内切酶进行线性化。这个步骤在32℃下在增加的pH下进行以改进酶的效率。在5小时之后,收获细菌并纯化MC载体。
随后,使用采用专门化ZYCY10P3S2T细菌菌株的改进系统(Kay等,2010,NatureBiotech 28:1287-89)。使用的表达盒是具有靶向突变β球蛋白内含子的反义序列的U7asLuc(Kang等,1998,Biochem 37:6235-39)和具有靶向mdx肌营养不良蛋白(dystrophin)前mRNA中跨越内含子22中剪接分支点的部分和内含子23中供体位点处的U1结合区的序列的U7asDys(Goyenvalle等,2004,Science 306:1796-99)。将经修饰U7基因连同它的天然启动子和3’元件一起克隆至pMC亲本质粒中的两个重组位点之间。在转导携带ΦC3整合酶和I-Sec-I核酸内切酶的诱导性基因的ZYCY10P3S2T细菌之后,根据由Kay等(2010,Nature Biotech 28:1287-89)发表的方法产生miMC。
所有使用的MC都在振荡孵育器中以小规模发酵产生。在用以在较大规模上产生MC的实验中,我们利用中试规模发酵罐。发酵罐是一种类型的生物反应器,其含有和控制微生物(在我们的情况下是细菌)的培养物。发酵罐含有合适的生长培养基,并且已用细菌培养物接种以使发酵过程开始。它控制培养的温度、pH、通气和搅拌。它也可通过使用在发酵过程期间连续或在设置时间点添加缓冲液的补料分批系统来设置以控制营养物的条件,例如通过添加阿拉伯糖来诱导重组。发酵使得达成高得多的细菌密度,以及因此实现较高质粒或MC DNA产率。
使用来自QIAGEN(Hilden,Germany)的商业试剂盒,根据制造商方案,采用溶解步骤需要较大缓冲液体积的修改,对MC DNA进行纯化。在纯化之后,使用在260nm下进行的紫外分光光度测定法以及琼脂糖凝胶电泳来测定数量和品质。
对于一些用途,重要的是仅具有一种存在于最终产物中的异构体,即无任何污染质粒的单体T20MC。因此,我们进行目标异构体的额外凝胶分离和纯化。在琼脂糖凝胶上,MCDNA根据尺寸被分离。应小心避免由紫外光或插入染料使DNA产生切口:在凝胶的染色边缘上标记异构体在凝胶中的位置,并且从未染色凝胶切除相应条带。接着通过QIAquick凝胶提取试剂盒(QIAGEN),根据制造商方案,从这个凝胶片段提取单体,并且进一步通过苯酚:氯仿提取加以纯化。
气动递送-通过使用(Bioject Medical Technologies Inc,CA,USA)穿过小鼠皮肤注射DNA载体来评价不同尺寸的MC经受由气动递送诱导的剪切力的能力。通过凝胶电泳来分析MC完整性。
是一种通过迫使液体药物穿过抵靠皮肤加以保持的微小孔口来用于药物递送的无针系统。气动以及孔口的小直径产生在不使用针的情况下穿透皮肤的超细高压流体流。可将注射液递送至各种深度,其中肌肉内注射是最深注射类型。大多数疫苗当前被递送至肌肉内深度。也可将药剂皮下递送至皮肤以下的脂肪层中,以及进行真皮内递送。在各种方法中,气动递送可用于向人受试者施用T20 MC。
脂质体转染- 6(Roche,Mannheim,Germany)用于根据制造商方案,以试剂:DNA比率3∶1使MC构建体脂质体转染至人纤维肉瘤细胞系HT-1080中。在转染之前一天以适当密度接种细胞以使它们在转染当天达到70-80%汇合。48小时后处理细胞。
在另一研究中,使用2000(ThermoScientific),根据制造商方案转染HeLa Luc/705细胞。简言之,在转染之前一天以适当密度接种细胞以使它们在转染当天达到70-80%汇合。使用每μg DNA 2.3μl2000,在OPTI-(ThermoScientific)中使DNA复合。在转染之后24小时处理细胞。脂质体转染是一种用于递送T20 MC来供人治疗使用的替代性技术。
电穿孔-也使用电穿孔来转染HeLa Luc/705细胞。电穿孔是一种物理转染方法,其使得能够直接递送越过细胞膜并进入核中。当将外部电场施加于细胞,并且跨膜电位超过临界阈值时,发生电穿孔(Golzio等,2004,Methods 33:126-35)。这导致可能通过产生纳米级孔隙而达成的使得将DNA递送至细胞中的质膜瞬时透化。我们使用转染系统(ThermoScientific),其中在产生电场所处的吸移器尖端腔室中悬浮处理细胞。
体内递送-将pDNA直接注射至肌肉中导致DNA在肌纤维细胞中表达。已在各种物种中证明在肌肉内注射裸露DNA之后,众多转基因得以摄取和表达(Braun,2008,Curr GeneTher 8:391-405)。表达在数天之后达到峰值,接着下降成较低但稳定表达,并且在一些情况下可在持续极其长久时间被检测到(Wolff等,1992,Hum Mol Gen 1:363-69)。然而,向骨骼肌中的pDNA基因转移的效率较低,其中在肌肉内注射之后约1%的细胞被转染(Jiao等,1992,Hum Gene Ther3:21-33)。此外,表达仅见于肌肉的极其受限区域中,通常沿着针道。在特定实施方案中,直接注射可用于施用T20MC供治疗使用。
流体动力学递送-流体动力学输注是一种证据充分的基因转移技术,已知在小鼠中会产生较高肝转染效率(Suda和Liu,2007,Mol Ther 15:2063-69)。通过快速注射相对大体积的DNA溶液,在毛细血管中产生受控流体动压,其增强细胞可渗透性并使得进入肝细胞中。肝中的不连续窦状毛细血管对流体动力学程序敏感,并且高压被认为会诱导肝细胞中的负责细胞内DNA转移的膜孔隙。当压力降低时,孔隙闭合,并且物质被圈闭在细胞内部(Zhang等,2004,Gene Ther 11:675-82;A1-Dosari等,2005,Adv Genetics 54:65-82)。
在示例性方法中,在麻醉下用2ml流体动力学输液通过尾部静脉处理小鼠,所述体积是小鼠体重的约10%。这提供体内长期表达。在一些实施方案中,流体动力学递送可用于采用T20MC的人疗法。
体内电穿孔-体内电转移基于将pDNA溶液注射至肌肉中,随后在组织上施加一系列电脉冲。已显示电穿孔不仅通过细胞透化,而且也通过对DNA分子的直接活性作用从而促进DNA迁移和细胞摄取来增加基因转移。已显示用牛透明质酸酶预处理肌肉会改善与电穿孔相关的细胞损伤(Gollins等,2003,Gene Ther 10:504-512)。这个方法据报道导致转导作用高于高压递送方法,产生的表达水平与用病毒载体实现的那些表达水平类似(Konieczny等,2013,Muscle&Nerve 47:649-63)。尽管电穿孔是一种具有相当侵袭性的基因递送方法,但研究已显示使用电穿孔在体内将非病毒载体递送至大鼠隔膜中是可行的(Beshay等,2009,Dev Growth Differ 51:547-53)。
进行电穿孔以增强将MC肌肉内注射至骨骼肌中的效率。根据Wells等(2008,Methods Mol Biol 423:421-31)对mdx小鼠进行透明质酸酶处理、注射和电穿孔。电穿孔可用于向受试者施用T20 MC供治疗使用,以改进细胞转导的效率。
实施例2.产生T20微环载体
我们将T20编码序列和用于HIV疫苗研究中的表达盒克隆至pMC载体中以产生小环。含有T20编码序列(以SEQ ID NO:2显示的氨基酸序列)的构建体由Immunomedics提供。在诱导重组之后,所得MC是1.1 kb,即小于相应常规质粒的三分之一。所得T20 MC载体的DNA序列以SEQ ID NO:1显示。
T20小、环序列
ACATTACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTACCCTGTTATCCCTAGATGACATT
ACCCTGTTATCCCTAGATGACATTTACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTACCC
TGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACCCTGTTATCCCTAGATGACATTACCCTGTTATC
CCAGATGACATTACCCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACCCTGTTATCCCTAGAT
GACATTACCCTGTTATCCCAGATGACATTACCCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATA
CCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTACCCTGTTATCCCTAGATACATTACCCTGT
TATCCCAGATGACATACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTACCCTGTTATCCCT
AGATACATTACCCTGTTATCCCAGATGACATACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGAC
ATTACCCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACCCTGTTATCCCTAGATGACATTACCC
TGTTATCCCAGATAAACTCAATGATGATGATGATGATGGTCGAGACTCAGCGGCCGCGGTGCCAGGGCGTGCC
CTTGGGCTCCCCGGGCGCGACTAGTGAATTCAGATCTGATATCTCTAGAGGCCCATTGCATACGTTGTATCCAT
ATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTA
ATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATG
GCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA
ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT
ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG
ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGG
CAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG
GGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATG
GGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA
CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCCGCGCACGGCAA
GAGGCGAGGGGCGGCGACTGAATTGGGTGTCGACCAGCCACCATGGAGACAGACACACTCCTGCTATGGGTA
CTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCTATACCAGCCTGATTCATAGCCTGATTGAAGAAAG
CCAGAACCAGCAGGAAAAAAACGAACAGGAACTGCTGGAACTGGATAAATGGGCGAGCCTGTGGAACTGGT
TTTGAGAATTCATCGAGTAACTATTGTGTCATGCAACATAAATAAACTTATTGTTTCAACACCTACTAATTGTGC
TGCAGGGGCCCGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGTAATCAGCATCATGATGTGGTA
CCACATCATGATGCTGATTATAAGAATGCGGCCGCCACACTCTAGTGGATCTCGAGTTAATAATTCAGAAGAAC
TCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCG
GTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCA
CACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATC
GCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGC
GAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGA
TGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGC
CATGATGGATACTTTCTCGGCAGGAGCAAGGTGTAGATGACATGGAGATCCTGCCCCGGCACTTCGCCCAATA
GCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCA
CGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGG
CGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGA
ATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCAT
CCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCGAGAAAGCCATCCAGTTTACTTT
GCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCC
AGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGA
TAGCCCAGTAGCTGACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGCTCGAGGGGGGCC
AAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGC
AGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACT
CAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCAT
CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCT
GAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGAC
GCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAA
ACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGACCAAAATCCCTTAACGTGAGTTTTCG
TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC
TGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCC
GAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC
TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGA
TAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG
GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGA
GAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG
AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT
TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA
CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTA
TTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG
AAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACT
CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATG
GCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA
GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCA
GCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCT
GCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTACATC
ATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGA
AATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCA
GCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATG
TGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGAT(SEQ ID NO:1)
T20
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF(SEQ ID NO:2)
使用转脂胺,根据制造商方案,将24μg载体添加至10cm板中,将MC转染至HeLa、Hek和U2OS细胞中。图l显示在转染之后48小时,免疫沉淀样品与粗细胞溶解产物两者中的ssT20体外表达。较大分子量的突出条带是用于免疫沉淀的抗体的重链和轻链。当相同抗体用于蛋白质印迹时,这些条带也通过二抗而检测到。这个研究显示在经转染人细胞中产生T20肽是可行的。
T20先前已被表达为多聚融合蛋白,显示所述融合蛋白在体外具有抗病毒作用(Hacein-Bey-Abina等,2008,J Clin Invest 118:3132-42)。Dervillez等(2006,Chem MedChem 1:330-39)也表达单独C46肽(一种T20源性肽),并且显示所述肽得以表达,但不分泌。然而,他们未研究他们的肽构建体的免疫原性。对于免疫刺激,分泌并不关键,因为经转染细胞可递呈在MHC分子上的抗原。因此,T20可被识别为外来,并且触发免疫应答。在此处,我们显示MC载体可在人细胞系中表达单体T20肽,并且蛋白质可通过使用中和人抗体2F5进行蛋白质印迹加以检测。
后续努力涉及改进MC产物的纯度。我们分析许多不同细菌菌株,并且发现使用以它的高蛋白质产生效率著称的O17大肠杆菌菌株导致重组和降解改进。我们也尝试使MC质粒与组成型表达阿拉伯糖输入作用的质粒共同发酵,但难以平衡两种质粒在细菌中的拷贝数比率。
为产生为临床使用所需的数量,方法需要从振荡孵育器按比例扩大至发酵罐规模。用5升培养基在(Falkenberg,Sweden)发酵罐中进行关于第一代MC系统的中试规模发酵实验。使亲本质粒生长过夜,接着通过注射阿拉伯糖来诱导小环形成。继续生长4小时。一个5升发酵批次产生65mg产物,但它具有极高含量的未重组和未消化质粒,仅含有15%MC。对MC产生系统的这个初始按比例扩大研究显示可获得高质粒产率,但需要改进以在最终产物中达到较高纯度。
在Kay等(2010,Nature Biotech 28:1287-89)公开他们的采用专门化ZYCY10P3S2T细菌菌株的改进系统之后,进一步研究利用在ZYCY10P3S2T中形成MC。使用这个系统,通过在发酵的诱导期期间在若干时间点添加L-阿拉伯糖以模拟补料分批发酵,小MC载体的产率得以改进。新的T20MC产生方法提供相对纯的T20MC的高产率。
实施例3.基于T20微环的疫苗
T20MC适用作用以抑制或预防HIV感染的疫苗系统。T20阻断病毒生命周期中的早期膜融合步骤,并且可预防重新感染以及细胞至细胞病毒传播。尽管先前已显示抗gp41抗体不损害T20在治疗HIV时的抗病毒作用,但关于T20自身的免疫保护性质所知甚少(Walmsley等,2003,J Infect Dis 188:1827-33)。T20肽可被识别为HIV抗原,因为它含有广泛反应性2F5表位(Muster等,1993,J Virol 67:6642-47)。T20肽的这个免疫反应性活性由所述肽模拟HIV-1糖蛋白gp41的残基643-678的事实所致。因此,针对T20肽形成的任何抗体或CTL反应性都也将结合HIV-1跨膜蛋白的这个关键区域。对gp41和gp120的天然抗体应答在HIV感染之后不久即发生,并且可产生中和抗体。
将T20肽作为HIV蛋白/DNA疫苗的一部分加以评价(图2)。图2显示连同HIV-1 DNA疫苗构建体一起施用T20肽使抗Env免疫原性增强。因此,我们将T20编码序列和先前用于HIV抑制研究中的表达盒(Calarota等,1998,Lancet 351:1320-25)克隆至pMC载体中以产生MC。含有T20编码序列的构建体如Chang等(2012,PLoS One 7:e41235)中所公开。
实施例4.使用DDD部分来增强免疫原性
在某些实施方案中,可通过施用T20或其它病毒抗原与一种或多种佐剂组合来增强诱导针对HIV感染的免疫应答的能力。通过增强全身性免疫应答,佐剂可促进对针对所靶向抗原诸如T20的特异性免疫应答的更有效诱导。
构建融合蛋白(由SEQ ID NO:3编码),其包含通过铰链接头(由SEQ ID NO:15编码)接合于T20序列(SEQ ID NO:2)的DDD2部分(SEQ ID NO:5),其中添加(His)6GS(SEQ IDNO:16)部分以用于亲和色谱法中。总体融合蛋白的结构显示于图3中。
含有DDD的构建体与T20融合蛋白在本文中被称为“T20质粒”以区别于不含有DDD的T20MC。进行证明抗原(例如T20)融合于DDD部分使DNA疫苗的用以在经免疫受试者中产生抗原特异性抗体的效能增强的实验。
尽管DDD使DNA疫苗的效能增强所采用的作用机理当前未知,但一种可能解释是在DC中表达的DDD-T20蛋白结合脂质筏中的AKAP,此有助于T20的抗原递呈,从而导致抗T20抗体显著增加。Schillace等(2009,PLoS One e4807;2011,Immunol Cell Biol 89:650-58)报道抗原递呈需要树突细胞(DC)AKAP,但不含有关于与DNA疫苗一起使用DDD-抗原融合蛋白来使抗原特异性抗体的产生增加的公开内容或暗示。作为替代,针对AKAP的抑制剂由Schillace用于阻止DC中的抗原递呈。
这个假设可用包含融合的DDD-X基因的各种DNA疫苗加以测试。X的示例性选择包括但不限于T20、CEACAM5的A3B3结构域、CD20的细胞外结构域、PD-L1和PD-1蛋白。所述DNA疫苗可通过将融合的DDD-X基因克隆至可商购获得的质粒pKCMV中来产生。主题DNA疫苗具有以下优势:成本低,容易构建,以及交叉递呈I类抗原与II类抗原两者。
尽管DDD2部分(SEQ ID NO:5)用于初始融合蛋白构建体,但可将其它已知DDD部分并入主题融合蛋白、表达载体和疫苗中。举例来说,DDD1(SEQ ID NO:4)也可被利用,并且即使不更有效,也可与DDD2同样有效。
以下显示DNA疫苗在BALB/C小鼠中的示例性免疫时程。术语“质粒Env亚型A、B和C”如Nilsson等(2015,PLoS ONE)中所公开,并且是指编码HIV-1基因gp160亚型A、B和C的DNA质粒。小环T20和质粒T20如上所公开。在0、4和8周使小鼠免疫。
BALB/C小鼠的免疫时程
A组:
●质粒Env亚型A、B和C(20μg)+小环T20(20μg)
B组:
●质粒Env亚型A、B和C(20μg)+质粒T20(20μg)
C组:
●质粒Env亚型A、B和C(20μg)+T20蛋白(10μg)
D组:
●质粒Env亚型A、B和C(20μg)+空pKCMV(20μg)
E组:
●空质粒pKCMV(40μg)
在如上所示加以免疫的A组小鼠与D组小鼠之间进行比较。图4显示在3次免疫之后,A组中的经免疫小鼠中抗T20抗体和抗gp140C抗体的产生。结果指示质粒Env+小环T20成功产生gp140C特异性抗体与T20特异性抗体两者,并且在第3次免疫之后,效价增加约5倍。图5显示免疫应答依赖于在经免疫小鼠中存在T20表达。将A组(质粒Env A、B和C+T20MC)相对于D组(质粒Env A、B和C+空pKCMV载体)中的经免疫小鼠关于在3次免疫之后,针对T20的抗体和针对gp140C的抗体的存在性进行比较。结果显示小环T20与质粒Env组合成功产生T20抗体,而单独质粒Env未能诱导此举。尽管D组中的小鼠产生低效价的针对gp140C的抗体,但当添加T20MC时,抗体产生水平得以实质上增加。
如上所概述进行另一实验以比较对针对T20的免疫应答和针对gp140C的免疫应答的诱导。观察到在比较A组(采用T20,无DDD2)相对于B组(采用T20,有DDD2)中的经免疫小鼠时,相较于在不存在DDD2下的相同免疫,以与T20的融合蛋白形式添加DDD2(SEQ ID NO:5)部分,疫苗产生高8倍的抗体效价水平。因此,将DDD2添加至T20MC疫苗中有效促进形成针对T20的抗体和针对gp140C的抗体。
实施例5.用T20MC构建体进行体内肽产生和基因疗法
用诸如VEGF的生长因子治疗缺血性组织已在动物实验中显示有前景的结果,但临床试验令人失望(Henry等,2003,Circulation 107:1359-65)。这部分地归因于生长因子在体内具有短暂半衰期。阻止基因疗法成功临床应用于心脏疾病的问题与低效基因转移、宿主免疫应答以及缺乏可持续治疗性转基因表达相关。
因为MC构建体解决这些问题中的至少两者,所以进行研究以将T20蛋白由MC载体的表达与由质粒构建体的表达进行比较。将用于临床前研究中的质粒载体与以上实施例2中公开的MC亲本质粒和T20MC构建体进行比较。通过直接肌肉内注射裸露DNA来递送构建体。与亲本质粒含有相同表达盒但仅涵盖1.1kbp的T20MC载体显示增加的体内转染和蛋白质表达,从而使得将T20MC用于HIV基因疗法。
这些结果与在其它模型系统的情况下对于基于MC的基因疗法报道的那些结果一致。Lijkwan等(2014,Hum Gene Ther 25:41-49)使用荧光素酶作为报告基因来将小鼠骨骼肌中由用MC和质粒构建体进行等摩尔治疗获得的表达进行比较。他们报道持续多达28天,由MC获得的表达显著更高。Huang等(2009,Circulation 130:S60-69)在鼠类心脏中进行类似比较。在心脏中,MC比质粒具有更高表达,并且荧光素酶持续90天可检测。当使用在治疗上更相关的HIF-1α蛋白时,Huang等(2009)能够在治疗之后14天检测到由MC载体获得的显著表达,其中水平是质粒构建体的水平的多于两倍。
尺寸较小、不存在抗生素抗性基因以及CpG含量较低使得MC成为适用于基因疗法治疗的替代方案。
实施例6.MC递送方法
MC构建体可通过不同替代性方法来递送以供治疗使用。我们的研究指示电穿孔可为一种适于使MC向宿主组织中的递送增强的方法。载体自身也可被修饰以通过电穿孔来递送。已被显示在电穿孔之后会增加平滑肌中基因表达的序列是平滑肌γ-肌动蛋白启动子的区域(Young等,2008,Exp Biol Med 233:840-48)。它含有驱动载体的核积累的组织特异性转录因子结合位点。
对下肢的流体动力学灌注是已成功用于向肌肉中递送病毒载体和ASO,以及已成功用于质粒的另一方法。然而,流体动力学和电穿孔都不将是用于遍及身体进行全身性递送的实际可行工具。这些方法将更适于局部递送载体以进行治疗。当利用T20MC例如作为疫苗系统来诱导针对T20,以及最终针对gp41和完整HIV或受HIV感染细胞的全身性免疫应答时,局部递送将有效用于免疫系统活化,此将接着提供针对HIV的全身性保护。经高效转染的肌肉在原则上也可用作分泌的治疗性蛋白质的产生部位,所述分泌的治疗性蛋白质可在身体中其它地方碰到它的靶标,诸如T20(恩夫韦地)。因为骨骼肌细胞不分裂,所以载体将长久存在于组织中。
流体动力学也已在若干研究中用于将MC递送至小鼠的肝中,并且已显示相较于质粒,表达更高和更稳健。超声是已用于靶向小鼠唾液腺的MC载体的基因递送的另一物理方法(Geguchadze等,2014,Meth Clin Dev 1:14007)。研究报道相较于常规质粒,MC载体具有增加的荧光素酶表达。相较于MC载体,用质粒转染导致尤其在与免疫性、细胞应激和形态发生相关的路径中蛋白质含量变化。当使用MC作为基因递送载体时,这些影响得以实质上降低。质粒骨架诱导与当使用病毒载体时所见的免疫应答类似的免疫应答。因此,相比于全长质粒载体,MC载体使得能够达成较高表达,并且毒性也较小。
化学递送是用以实现更高效递送非病毒载体的另一方法。化学递送是指使用不同载体脂质或肽来紧缩和保护DNA并增强细胞摄取。存在关于使载体的化学性质优化,以及使它偶联于信号部分以达成改进摄取和细胞内移行的众多研究。这些可为核定位信号或细胞结合配体。所述配体也可促进载体靶向给定器官。使用共价或非共价连接于运载物的细胞渗透肽可有助于进入细胞中。作为可如何组合这些方法的一实例,Ko等(2009,Gene Ther16:52-59)报道当用细胞渗透肽和对心脏肌球蛋白具有特异性的单克隆抗体修饰pDNA和脂质复合物时,在大鼠中通过静脉内注射达成的靶向缺血性心肌的基因递送得以增强。
实施例7.DNA疫苗接种
由于全身性递送效率的限制,所以T20MC可能在仅需要局部表达时更适于使用。一种所述情况是DNA疫苗接种,其中通常将抗原真皮内或肌肉内递送至小区域中。已显示电穿孔是一种用于在人中引发强烈免疫应答的高效递送方法(Sallberg等,2015,MedMicrobiol Immunol 204:131-35)。据信DNA摄取增加与充当辅佐因素的局部组织损害两者均会使免疫改进。如上所讨论,由于T20MC的尺寸较小,所以相比于常规质粒,它可为用于用电穿孔进行递送的更加最优载体。尺寸较小的另一益处是相较于正常质粒,当使用MC时,每μg DNA的治疗剂量增加。这具有重要性,因为高局部转基因表达对于达成良好免疫应答至关重要。
被认为会诱导免疫应答的质粒DNA的一个特征是具有高含量的未甲基化CpG,其可充当疫苗的佐剂。相比于常规质粒,用于DNA疫苗接种的MC载体由于它的尺寸较小而将具有较少CpG。然而,可将优化的CpG序列克隆至MC盒中。Coban等(2005,J Leuk Biol 78:647-55)已显示在质粒中包括某些优化的CpG序列使小鼠中的免疫应答增强。这些序列仅在30与50bp之间,因此所得MC将仍然显著小于常规质粒。或者,可将MC连同合成CpG寡核苷酸一起递送。与多种疫苗药剂一起共同施用CpG寡核苷酸已改进体液和细胞免疫应答(Klinman等,2009,Adv Drug Delivery Rev 61:248-55)。出于DNA疫苗目的,可极其充分有利的是不使佐剂共价连接于将由其表达抗原的DNA序列。
实施例8.离体疗法
离体疗法可被视为局部治疗性基因表达的一实例。离体治疗旨在从患者移除细胞(例如树突细胞),在实验室中对它们进行修饰,接着使经修饰细胞返回至所述患者中,在所述患者中,它们使疾病受影响。对于离体使用,可有益的是使用高治疗剂量的缺乏任何抗性基因,可被转移回患者中的非整合载体。Evans和Hyde(2015,Rheumatology 11:234-42)综述用以使肌肉骨骼系统再生的若干基因治疗方法,在它们之中的是主要使用病毒载体的离体策略。Usas等(2007,Biomaterials 28:5401-06)研究肌肉源性干细胞和它们的离体修饰,例如使用逆转录病毒载体来增强骨形成。他们也用增强骨产生的生长因子诸如VEGF的组合修饰干细胞。Sheyn等(2008,Stem Cells 26:1056-64)使用用质粒载体离体修饰的脂肪组织源性干细胞来增强骨形成和脊柱融合。他们推断用于离体对干细胞进行遗传工程改造的非病毒基因递送方法是安全和瞬时的,从而将成骨性基因的过表达限于数周的时期。MC载体也已离体用于人脂肪源性基质细胞中以诱导Bcl-2促存活蛋白的瞬时过表达(Hyun等,2013,Stem Cells Transl Med 2:690-702)。这在植入小鼠模型中后使细胞存活和骨形成增强,并且持续多达4周观察到表达。
在某些实施方案中,诸如对于诱导针对HIV的宿主免疫,将T20MC离体递送至抗原递呈细胞诸如树突细胞中可用于实施所要求保护的方法。
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本文公开和要求保护的所有组合物和方法都可鉴于本公开在不进行过度实验下制备和执行。尽管组合物和方法已关于优选实施方案加以描述,但为本领域技术人员显而易知的是可在不脱离本发明的概念、精神和范围下向组合物和方法以及在本文所述的方法的步骤方面或在步骤的顺序方面应用变化。更具体来说,在化学与生理两方面均相关的某些试剂可替代本文所述的试剂,同时将实现相同或类似结果。为本领域技术人员显而易知的所有所述类似替代物和修改都被视为在如由随附权利要求限定的本发明的精神、范围和构思内。
序列表
<110> 免疫医疗公司
<120> 用于抗HIV (人免疫缺陷病毒)疗法和/或疫苗的T20构建体
<130> IMM360WO1
<140>
<141>
<150> 62/167,404
<151> 2015-05-28
<160> 16
<170> PatentIn 3.5版
<210> 1
<211> 5132
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述:合成多核苷酸
<400> 1
acattaccct gttatcccta gatgacatta ccctgttatc ccagatgaca ttaccctgtt 60
atccctagat gacattaccc tgttatccct agatgacatt taccctgtta tccctagatg 120
acattaccct gttatcccag atgacattac cctgttatcc ctagatacat taccctgtta 180
tcccagatga cataccctgt tatccctaga tgacattacc ctgttatccc agatgacatt 240
accctgttat ccctagatac attaccctgt tatcccagat gacataccct gttatcccta 300
gatgacatta ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct 360
gttatcccag atgacatacc ctgttatccc tagatgacat taccctgtta tcccagatga 420
cattaccctg ttatccctag atacattacc ctgttatccc agatgacata ccctgttatc 480
cctagatgac attaccctgt tatcccagat gacattaccc tgttatccct agatacatta 540
ccctgttatc ccagatgaca taccctgtta tccctagatg acattaccct gttatcccag 600
atgacattac cctgttatcc ctagatacat taccctgtta tcccagatga cataccctgt 660
tatccctaga tgacattacc ctgttatccc agataaactc aatgatgatg atgatgatgg 720
tcgagactca gcggccgcgg tgccagggcg tgcccttggg ctccccgggc gcgactagtg 780
aattcagatc tgatatctct agaggcccat tgcatacgtt gtatccatat cataatatgt 840
acatttatat tggctcatgt ccaacattac cgccatgttg acattgatta ttgactagtt 900
attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 960
cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 1020
caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 1080
tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta 1140
cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 1200
ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 1260
tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 1320
caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 1380
ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 1440
gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc 1500
cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgc ggcccgcgca 1560
cggcaagagg cgaggggcgg cgactgaatt gggtgtcgac cagccaccat ggagacagac 1620
acactcctgc tatgggtact gctgctctgg gttccaggtt ccactggtga cgcggcctat 1680
accagcctga ttcatagcct gattgaagaa agccagaacc agcaggaaaa aaacgaacag 1740
gaactgctgg aactggataa atgggcgagc ctgtggaact ggttttgaga attcatcgag 1800
taactattgt gtcatgcaac ataaataaac ttattgtttc aacacctact aattgtgctg 1860
caggggcccg ccccaactgg ggtaaccttt gagttctctc agttgggggt aatcagcatc 1920
atgatgtggt accacatcat gatgctgatt ataagaatgc ggccgccaca ctctagtgga 1980
tctcgagtta ataattcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga 2040
atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc 2100
ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg 2160
gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc 2220
atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg ctcgccttga gcctggcgaa 2280
cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc 2340
ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca 2400
ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc 2460
ggcaggagca aggtgtagat gacatggaga tcctgccccg gcacttcgcc caatagcagc 2520
cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg 2580
gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg 2640
gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 2700
cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 2760
gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga 2820
tcagagcttg atcccctgcg ccatcagatc cttggcggcg agaaagccat ccagtttact 2880
ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct 2940
gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt 3000
ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt 3060
cagcaccgtt tctgcggact ggctttctac gtgctcgagg ggggccaaac ggtctccagc 3120
ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca 3180
gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc 3240
ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg 3300
cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc 3360
tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga 3420
gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa 3480
actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta 3540
caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaccaaaat 3600
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3660
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3720
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3780
cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 3840
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 3900
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 3960
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4020
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4080
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4140
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4200
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4260
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4320
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4380
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 4440
gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 4500
cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 4560
gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 4620
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 4680
cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag 4740
gcgaagcggc atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta 4800
tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct 4860
acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat 4920
tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg 4980
gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg 5040
cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc 5100
gacaagcaaa catgctgtgc gacgctggcg at 5132
<210> 2
<211> 36
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 2
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30
Trp Asn Trp Phe
35
<210> 3
<211> 472
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述:合成多核苷酸
<400> 3
ggcggccaca tccagatccc gccggggctc acggagctgc tgcagggcta cacggtggag 60
gtgctgcgac agcagccgcc tgacctcgtc gaattcgcag tggagtactt cacccgcctg 120
agagaagctc gcgctgagtt ccctaaaccc agcactccac ccggatcttc cggccaccac 180
caccaccacc acggatccta taccagcctg attcatagcc tgattgaaga aagccagaac 240
cagcaggaaa aaaacgaaca ggaactgctg gaactggata aatgggcgag cctgtggaac 300
tggttttgac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga 360
aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 420
tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at 472
<210> 4
<211> 44
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 4
Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr
1 5 10 15
Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala
20 25 30
Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala
35 40
<210> 5
<211> 45
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 5
Cys Gly His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly
1 5 10 15
Tyr Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
20 25 30
Ala Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala
35 40 45
<210> 6
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成肽
<400> 6
Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln
1 5 10 15
Ala
<210> 7
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成肽
<400> 7
Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile
1 5 10 15
Gln Gln Ala Gly Cys
20
<210> 8
<211> 50
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 8
Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys His Asn Ile Gln Ala
1 5 10 15
Leu Leu Lys Asp Ser Ile Val Gln Leu Cys Thr Ala Arg Pro Glu Arg
20 25 30
Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Arg Leu Glu Lys Glu Glu
35 40 45
Ala Lys
50
<210> 9
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 9
Met Ser Cys Gly Gly Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys
1 5 10 15
His Asn Ile Gln Ala Leu Leu Lys Asp Ser Ile Val Gln Leu Cys Thr
20 25 30
Ala Arg Pro Glu Arg Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Arg
35 40 45
Leu Glu Lys Glu Glu Ala Lys
50 55
<210> 10
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成肽
<400> 10
Cys Gly Phe Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser
1 5 10 15
Asp Val Phe Gln Gln Gly Cys
20
<210> 11
<211> 51
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 11
Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys His Asn Ile Gln Ala
1 5 10 15
Leu Leu Lys Asp Val Ser Ile Val Gln Leu Cys Thr Ala Arg Pro Glu
20 25 30
Arg Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Lys Leu Glu Lys Glu
35 40 45
Glu Ala Lys
50
<210> 12
<211> 54
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 12
Ser Leu Lys Gly Cys Glu Leu Tyr Val Gln Leu His Gly Ile Gln Gln
1 5 10 15
Val Leu Lys Asp Cys Ile Val His Leu Cys Ile Ser Lys Pro Glu Arg
20 25 30
Pro Met Lys Phe Leu Arg Glu His Phe Glu Lys Leu Glu Lys Glu Glu
35 40 45
Asn Arg Gln Ile Leu Ala
50
<210> 13
<211> 44
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 13
Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr
1 5 10 15
Thr Val Glu Val Gly Gln Gln Pro Pro Asp Leu Val Asp Phe Ala Val
20 25 30
Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Arg Gln
35 40
<210> 14
<211> 44
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成多肽
<400> 14
Ser Ile Glu Ile Pro Ala Gly Leu Thr Glu Leu Leu Gln Gly Phe Thr
1 5 10 15
Val Glu Val Leu Arg His Gln Pro Ala Asp Leu Leu Glu Phe Ala Leu
20 25 30
Gln His Phe Thr Arg Leu Gln Gln Glu Asn Glu Arg
35 40
<210> 15
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述:合成寡核苷酸
<400> 15
gagttcccta aacccagcac tccacccgga tcttccggc 39
<210> 16
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:合成肽
<400> 16
His His His His His His Gly Ser
1 5
Claims (19)
1.一种表达T20的经分离核酸,所述经分离核酸的核苷酸序列如SEQ ID NO:1所示。
2.一种T20表达载体,其包含SEQ ID NO:1的核苷酸序列。
3.如权利要求2所述的表达载体,其中所述表达载体是小环载体。
4.一种疫苗,其包含根据权利要求2所述的T20表达载体。
5.一种药物组合物,其包含根据权利要求2所述的T20表达载体。
6.根据权利要求2所述的T20表达载体在制备转染人树突细胞(DC)的制剂中的用途,其包括:
a) 获得人树突细胞(DC);
b) 用根据权利要求2所述的T20表达载体转染所述人DC;以及
c) 向人受试者施用经转染的所述人DC,
其中经转染的所述人DC抑制所述受试者的HIV感染,或经转染的所述人DC预防所述受试者的HIV感染。
7.如权利要求6所述的用途,其中所述人DC是从所述人受试者分离的自体DC。
8.如权利要求6所述的用途,其中所述人DC是同种异体DC。
9.如权利要求6所述的用途,其进一步包括使所述人DC暴露于选自由脂多糖、GM-CSF和干扰素-α组成的组的辅助化合物。
10.如权利要求6所述的用途,其进一步包括向所述受试者施用至少一种抗HIV剂。
11.如权利要求10所述的用途,其中所述抗HIV剂选自由HAART、逆转录酶抑制剂、融合抑制剂、蛋白酶抑制剂、整合酶抑制剂和抗HIV抗体组成的组。
12.如权利要求10所述的用途,其中所述抗HIV剂选自由以下组成的组:4,4-二氟-N-((1S)-3-(外型-3-(3-异丙基-5-甲基-4H-1,2,4-三唑-4-基)-8-氮杂双环(3.2.1)辛-8-基)-1-苯基丙基)环己烷甲酰胺、阿巴卡韦、氨多索韦、安普那韦、阿立他滨、阿扎那韦、贝韦立马、BMS-378806、BMS-488043、卡拉诺利A、塞拉集宁、可比司他、蓝藻抗病毒蛋白-N、地瑞那韦、二芳基嘧啶、地达诺新、度鲁特韦、依法韦仑、埃替格韦、艾夫他滨、恩曲他滨、恩夫韦地、没食子酸表没食子儿茶素、依曲韦林、费替那韦、福沙那韦、膦甲酸、格瑞弗森、格鲁博南A、羟基脲、茚地那韦、拉米夫定、来非那韦、来司韦林、洛匹那韦、马拉韦罗、米替福新、MK-2048、奈非那韦、奈韦拉平、普乐沙福、拉西韦、雷特格韦、利匹韦林、利托那韦、沙奎那韦、塞利西利、司他福定、司他匹定、司他夫定、T20、TAK 779、TAK-220、Tat拮抗剂、替诺福韦、替拉那韦、天花粉蛋白、维克维洛、扎西他滨和齐多夫定。
13.如权利要求11所述的用途,其中所述抗HIV抗体是伊巴珠单抗。
14.权利要求2的T20表达载体在制备转染人受试者中的细胞的制剂中的用途,其包括:
a) 向人受试者施用所述T20表达载体;以及
b) 用所述T20表达载体转染所述人受试者中的细胞;
其中经转染的所述细胞表达T20肽,
其中所述T20肽抑制所述受试者的HIV感染,或所述T20肽预防所述受试者的HIV感染,
其中所述T20肽的氨基酸序列是SEQ ID NO: 2。
15.如权利要求14所述的用途,其中所述T20肽被分泌至所述受试者的循环中。
16.如权利要求14所述的用途,其中经转染的所述细胞诱导所述受试者中针对T20、gp41和HIV的免疫应答。
17.如权利要求16所述的用途,其中所述免疫应答有效预防所述受试者的HIV感染。
18.如权利要求16所述的用途,其中所述免疫应答有效杀灭所述受试者中受HIV感染细胞。
19.一种产生T20肽的方法,其包括:
a) 获得根据权利要求2所述的T20表达载体;
b) 用所述T20表达载体转染细胞系;以及
c) 培养经转染细胞以产生T20肽,
其中所述T20肽的氨基酸序列是SEQ ID NO: 2。
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US9993547B2 (en) | 2018-06-12 |
US20170246291A1 (en) | 2017-08-31 |
CA2982376A1 (en) | 2016-12-01 |
US20160346380A1 (en) | 2016-12-01 |
EP3303370A4 (en) | 2019-03-13 |
EP3303370A1 (en) | 2018-04-11 |
WO2016191481A1 (en) | 2016-12-01 |
CN107614515A (zh) | 2018-01-19 |
US9687547B2 (en) | 2017-06-27 |
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