CN101896601A - 用于生成树突细胞的方法 - Google Patents
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Abstract
本发明涉及一种用于生成树突细胞的方法及其在药物中的用途,这通过目的在于在功能上改善其在治疗癌症、微生物感染、变态反应、自身免疫性疾病或器官和干细胞移植排斥中的治疗功效的遗传工程来实现。
Description
发明领域
本发明涉及一种用于生成和遗传工程化改造树突细胞(DC)的方法及其用途。
发明背景
在过去多年期间,已经认可树突细胞(DC)为免疫的中心调节物。人DC是通过在存在生长因子(通常为白介素(IL)4和粒细胞-巨噬细胞集落刺激因子(GM-CSF))的情况中从造血干细胞或外周血单核细胞进行的体外分化生成的。最近的证据提示,DC具有柔性地(flexibly)响应微生物、外伤、或代谢应激遭遇的能力。如此,DC不仅分化成能履行特定功能(例如活化或耐受性,1型或2型T辅助淋巴细胞(Th1,Th2)极化)的一种亚型,而且还以适合于给定环境中遭遇的考验的时间-动力学方式呈现独特的功能状态(图1)。
单核细胞离开血流而进入各种组织,并变为通常称为未成熟DC(iDC)的细胞。这些iDC是通过从细胞外液以及来自生理学濒死细胞的凋亡体吸收材料来对其环境取样,进行加工,并在不进行共刺激的情况中以耐受性诱导形式将此材料呈递给T淋巴细胞的哨兵(sentinel)。可以认为耐受性诱导iDC表型是DC的缺省状态。此状态得到维持,直至iDC遭遇危险信号,该危险信号可以是由toll样受体(TLR)传送的病原体相关分子样式(PAMP)、炎性细胞因子、或T淋巴细胞衍生的信号传导(最主要由CD40/CD40L相互作用介导)。此过程称为DC成熟,其与功能改变的顺序一致。这些功能改变在一段约2天的时间里发生,之后DC达到称为成熟DC(mDC)的状态。最显著地,DC开始上调共刺激分子诸如B7家族成员CD80和CD86。这使DC能够向携带能与抗原性肽相互作用的T细胞受体的T淋巴细胞投递活化性而非抑制性信号,所述抗原性肽与DC膜上的主要组织相容性复合体(MHC)分子复合。在此阶段,还发生刺激物依赖性极化,其中DC分泌IL-12以及IL-12家族细胞因子,其有利于1型免疫应答,随后支持由细胞毒性T淋巴细胞(CTL)介导的细胞免疫。IL-12分泌一般由TLR与其配体结合(例如TLR4与LPS结合)触发,但是也通过可溶性或细胞膜结合的CD40L分子与DC上的CD40的相互作用触发。比较而言,IL-12释放的缺乏触发2型极化,其通过支持B淋巴细胞来启动体液免疫应答。在没有IL-12分泌的情况中启动DC成熟是通过iDC与细胞因子混合物(通常含有TNF-α和PG-E2以及多种炎性细胞因子,包括但不限于I型和II型干扰素、IL-1、或IL-6)的暴露实现的。
IL-12释放在约24小时后停止,指明DC与T淋巴细胞之间的相遇需要在该时间窗内发生以容许有效的1型极化和CTL活化。比较而言,共刺激分子的表达在2天后达到其最大值。因为按照定义,成熟的DC仅在表型方面而不在功能方面以共刺激分子的最大限度表达表征,所以释放IL-12的1型极化DC称为半成熟的(sm)DC。
约2天后,DC达到所谓成熟的阶段。它分化的第二天期间,DC免除其免疫刺激能力,并通过上调介导负调节反馈环的分子来获得免疫抑制特性(图1)。此分化阶段的生物学意义是在严格控制下保持免疫应答的必然性。活化的免疫细胞(特别是实现杀死其它细胞的CTL)对生物体形成相当大的威胁。这以避开其控制的免疫应答的病理学后果:自身免疫性疾病诸如I型糖尿病或多发性硬化例示。因此,遇到成熟信号后第1天期间引发免疫应答的同一DC会在其分化过程的第2天期间使此相同的免疫应答衰减。因此,成熟的DC实际上并不是如最初认为的免疫刺激细胞而是免疫抑制细胞,因此不足以进行目的在于免疫刺激的治疗性干预,诸如其在癌症免疫疗法或治疗微生物疾病中的用途。
辨别未成熟的(耐受性维持)、半成熟的DC(免疫刺激性)、和成熟的(免疫抑制性)DC是重要的(图1)。如上文所概述的iDC维持针对自身抗原的耐受性。smDC已经遇到上文所述成熟刺激物之一,而且已经不可逆地在约2天内分化成mDC。重要的是,仅在那2天的第一天期间,其实现IL-12释放,启动I型免疫极化,和随后支持CTL介导的免疫应答。一旦成熟的DC在一天后进入分化的第二阶段,它便获得免疫抑制特性。免疫学家能常规地通过膜分子诸如CD80、CD83、或CD86的表达来表征mDC。然而,与在几小时内达到最大限度表达,并在24小时后丧失的IL-12相比,这些膜分子仅在48小时后达到其最大限度表达。为了清楚地辨别本文中所描述的IL-12分泌DC与以名称成熟DC常规了解的细胞,选择术语半成熟的DC。非常重要的是,这不应暗示一些种类的功能缺陷,而仅仅是图1中按时间动力学比例的某个分化阶段。smDC在功能上与iDC以及mDC有所不同。
WO 2007/117682涉及用编码CD40L的mRNA分子转染的成熟树突细胞。
Koya R.C.等(J Immunoth.26(2003):451-460)描述了用编码CD40L的病毒转染未成熟的树突细胞。需要CD40L来使这些树突细胞成熟。
在Liu Y.等(Cancer Gene Therapy 9(2002):202-208)中,披露了用编码CD40L的病毒转染未成熟的树突细胞。
本发明的目的在于提供一种基于遗传工程来生成树突细胞的方法。可以使用这些树突细胞来制备药物制剂。
本发明涉及一种包含部分成熟的树突细胞的药物制剂,其通过包括下列步骤的方法可获得:
a)提供未成熟的树突细胞或其前体细胞或部分成熟的树突细胞,所述部分成熟的树突细胞如下可获得,即使未成熟的树突细胞与至少一种树突细胞成熟剂接触来生成部分成熟的树突细胞(半成熟的DC,smDC),如以其分泌IL-12的能力所定义的
b)操作步骤a)的所述细胞,特别是步骤a)的释放IL-12的所述部分成熟的树突细胞(半成熟的DC,smDC),以便
(i)过表达能够使以连续的IL-12分泌表征的树突细胞的T淋巴细胞刺激能力,并且选自CD40L的至少一种免疫分子维持至少24小时,优选至少48小时,其通过导入编码所述至少一种分子的核酸分子来实现;和/或
(ii)抑制或阻止在暴露于初级成熟剂诸如LPS/IFN-γ的树突细胞内起作用或自暴露于初级成熟剂诸如LPS/IFN-γ的树突细胞释放,并选自白介素10(IL-10)和吲哚胺2,3-加双氧酶(IDO)的至少一种T-淋巴细胞抑制分子,诸如表3、4和/或5中给出的至少一种基因的表达,其如下实现,即敲除表达所述至少一种T-淋巴细胞抑制分子的基因或其片段或导入用以抑制或阻止在所述树突细胞内有活性或从所述树突细胞投递至T细胞的至少一种T-淋巴细胞抑制分子的表达的核酸分子,优选核糖核酸分子,并
c)任选地,添加物质来将树突细胞的前体细胞转化成树突细胞。
依照本发明的药物制剂包含通过本文中所公开的方法可获得的树突细胞。进行目的在于过表达促成免疫刺激的分子诸如CD40L的遗传工程,或目的在于敲除免疫抑制分子诸如IL-10或IDO,和下文表3、4和5中所列新鉴定分子(其在DC中显示与IL-10和IDO类似的表达动力学)表达的遗传工程的树突细胞。可以对任何DC或前体细胞(如造血干细胞)实施遗传工程,不管这些树突细胞是暴露于成熟剂诸如TLR配体、炎性细胞因子的混合物、或T细胞衍生的信号诸如CD40L介导的信号,还是进行目的在于触发从未成熟的向成熟的DC进行表型转换的另一种规程,或者它们可以处于未成熟阶段。可以在暴露于成熟刺激物前或后实施(基因)操作。优选地,应用成熟刺激物(或成熟刺激物的组合)仅持续一段短暂的时间,例如不长于24小时,12小时,但是特别优选6小时,而且小于6小时。短暂的(至少2小时)成熟刺激物向DC的有利应用确保接种入患者中后的DC免疫药物保留了高效率启动T细胞刺激的能力。对DC的遗传工程化改造目的在于改善该基本免疫刺激能力,而并不意图替换它。
必须将用于生成本发明药物制剂的树突细胞的树突细胞前体细胞转化成树突细胞。其实现手段和方法是本领域已知的。
“树突细胞前体细胞”包括单核细胞、造血细胞等。
本发明的另一方面涉及一种用于生成树突细胞的方法,包括下列步骤:
a)提供未成熟的树突细胞,
b)使所述未成熟的树突细胞与至少一种树突细胞成熟剂接触来生成部分成熟的树突细胞(半成熟的DC,smDC),如以其分泌IL-12的能力所定义的,并
c)操作步骤b)的释放IL-12的所述部分成熟的树突细胞(半成熟的DC,smDC),以便
(i)过表达能够使以连续的IL-12分泌表征的树突细胞的T淋巴细胞刺激能力维持至少24小时,优选至少48小时或更长的至少一种免疫分子,并且所述至少一种免疫分子选自CD40L,其通过导入编码所述至少一种分子的核酸分子来实现;和/或
(ii)抑制或阻止在暴露于初级成熟剂诸如LPS/IFN-γ的所述树突细胞内起作用或自暴露于初级成熟剂诸如LPS/IFN-γ的树突细胞释放,并且选自白介素10(IL-10)和吲哚胺2,3-加双氧酶(IDO)的至少一种T-淋巴细胞抑制分子,诸如表3和4中给出的至少一种基因的表达,其如下实现,即敲除表达所述至少一种T-淋巴细胞抑制分子的基因或其片段或导入用以抑制或阻止在所述树突细胞内有活性或从所述树突细胞投递至T细胞的至少一种T-淋巴细胞抑制分子的表达的核酸分子,优选核糖核酸分子。
上文所述方法的步骤b)的经遗传工程化改造的IL-12释放DC过表达能够延长T淋巴细胞刺激时间窗和/或完全阻止其在24小时后关闭的至少一种分子,其特征在于使分泌维持得长于24小时,这通过导入编码所述至少一种免疫刺激分子的核酸分子来实现;或者显示暴露于任何有效的成熟刺激物后从T淋巴细胞刺激时间窗进入成熟后24小时开始打开的T淋巴细胞抑制时间窗的DC正常发育过程中所牵涉的至少一种分子的表达受到抑制或下调;和/或抑制或阻止已经发育成呈现免疫抑制表型的DC表达在介导T淋巴细胞抑制方面履行功能的至少一种分子。这是如下实现的,即敲除编码所述至少一种分子的基因或其片段和/或导入用以抑制或阻止干扰暴露于成熟刺激物后从免疫刺激性向免疫抑制性表型的DC正常发育的至少一种分子的表达的核酸分子,优选核糖核酸分子;和/或干扰从DC向T细胞投递的信号,引起抑制此T细胞的活性,如此抑制免疫应答。本发明的树突细胞使T淋巴细胞刺激(特别是CTL活化)最大化,其通过使用遗传工程来加宽约24小时的刺激时间窗或完全阻止24小时后此刺激时间窗的关闭。作为IL-10或IDO的备选,可以使用暴露于任何成熟刺激物后约24小时开始的DC免疫抑制功能中所牵涉的其它分子(表3、4、和5)。在制备经遗传工程化改造的免疫刺激性DC中优先靶向的会正是这些分子。特别优选的是,使用在所呈现的DNA微阵列数据中显示两倍过表达(表3和4),更优选至少六倍过表达的分子。表3和4中给出的数目显示了倍数过表达,如各自栏的标题中所标示的。特别优选的是,敲除表现出牵涉免疫抑制的分子在DC免疫药物中的表达,如图9中所描绘的例子所表明的。
通过反转上文所概述的策略,有可能设计经遗传工程化改造的T淋巴细胞抑制性DC免疫药物,用于在例如变态反应或自身免疫性疾病,以及干细胞和器官移植中治疗免疫系统的病理学反应过度。免疫抑制在生理学上是由在暴露于任何成熟刺激物后已经分化超过24小时的DC介导的。如下增强此类DC的免疫抑制能力,即干扰DC分化的前24小时期间的T淋巴细胞刺激分子的表达;和/或过表达赋予T淋巴细胞抑制的分子,其通过依照上文所概述的策略遗传工程化改造DC来实现。
smDC作为基因操作靶物的用途是本发明中心的且至关重要的部分。过去发表的mDC的免疫刺激性效果主要由于实施许多这些实验的高度人为的实验背景,例如使用本质上不存在的合成肽替换DC的真实靶物:天然的蛋白质抗原分子或者甚至全细胞,这两者都需要完全不同的DC摄取和加工的机制。许多其他调查人员使用鼠系统来进行他们的研究,而且人与小鼠之间有引起大量混淆的至关重要的差异。然而,现在普遍接受的是,mDC具有免疫抑制特性。令人惊讶的是,证明了用依照本发明的方法获得的树突细胞展现出更宽的刺激窗(即IL-12表达的升高和延长)。发现将半成熟(sm)DC-即如下的DC,其中生理学分化过程通过暴露于能够触发自DC进行IL-12分泌,但是不过优选在2至12小时后,更优选在6小时后消除的任何成熟刺激物启动-遗传工程化改造成过表达CD40L分子具有将其T淋巴细胞刺激能力维持至少24小时,优选48小时,而且甚至达5天或甚至10天的能力。此外,优选的是,在含有IFN-γ的培养基中培养此类经遗传工程化改造的DC。此类DC通过通常6小时暴露于Toll样受体(TLR)配体,优选但不排他的是脂多糖(LPS),再次优选在存在IFN-γ的情况中-参见表1-并遗传工程化改造成过表达CD40L来给予smDC,呈现如下表型,其特征在于连续的IL-12分泌持续至少1天,优选3天,且甚至达5天,而且在异基因混合白细胞反应(alloMLR)中的免疫刺激能力维持至少24小时,优选48小时,但是多至5天。应用于DC免疫药物设计,这确认DC分化的早期免疫刺激窗和较晚的免疫抑制窗的存在及相关功能。因此,开发刺激性DC免疫药物的一般原则可以加宽早期免疫刺激窗,以便更有效地触发免疫活化,并降低或关闭较晚的免疫抑制窗,或者对于设计抑制性DC免疫药物反之亦然(图1)。
一般而言,为了生成免疫刺激性DC药物(“DC免疫药物”;“免疫药物”)来治疗例如癌症或传染病,初始的成熟刺激物诸如LPS/IFN-γ需要被应用于DC,以便启动从iDC成为smDC的生理学分化。其它TLR配体(表1)可以达到与LPS相同的目的;TLR配体的组合可以给予更强的但没有性质差异的信号。若T淋巴细胞刺激性DC免疫药物的刺激潜力仅基于基因转移的人工操作而没有初始暴露于TLR配体介导的成熟刺激物(例如通过对未成熟DC的直接遗传工程化改造),则会丧失对DC功能的重要作用,并且T淋巴细胞刺激性DC免疫药物不能达到其完全的潜力。依照本发明的经遗传工程化改造的DC免疫药物与通过仅暴露于成熟剂或其组合(例如LPS/IFN-γ(smDC))制备的DC免疫药物的重要差异在于对于后一种,在DC分化相应的短暂窗期间应用smDC免疫药物是至关重要的。因此,此类刺激性DC免疫药物不得不在暴露于成熟刺激物后不久便进行应用,而遗传工程(例如通过过表达CD40L)目的在于加宽DC分化的免疫刺激时间,这容许应用时间的重要程度降低,但是最重要的是,阻止DC从免疫刺激性向免疫抑制性表型的发育(图1)。可以如下实现对DC的免疫刺激能力的相当大的改善,即敲除怀疑为免疫抑制中有重要牵涉的分子,如通过与已知的免疫抑制分子IL-10或IDO表达类似的表达谱(expression profile)所指明的(图3、4和5中所列的);或者已经显示为牵涉免疫抑制的分子,因为在DC中将它们敲除导致工程化改造DC的T细胞刺激能力改善(图9)。还有,较旧的smDC免疫药物的免疫刺激时间窗在24小时后关闭,而新型的经遗传工程化改造的DC免疫药物会使其T淋巴细胞刺激潜力再维持至少1天,优选3天,但多至5天。相当的思想适用于T淋巴细胞抑制性DC免疫药物。未成熟的DC首先需要被暴露于常规的成熟刺激物,诸如LPS/IFN-γ,以便启动朝向与DC分化的T淋巴细胞抑制窗对应的mDC表型的分化。可以在未成熟的DC成熟前通过成熟刺激物诸如LPS/IFN-γ,而且在靶向DC前体细胞诸如来自外周血的单核细胞,或造血干细胞和前体细胞时,特别但不排他的是在使用导致稳定整合入基因组中的基因转移方法诸如逆转录病毒基因转移时完成从DC免疫药物遗传工程化改造成过表达T-淋巴细胞抑制分子。在暴露于成熟刺激物前进行的遗传工程外,可以在通过例如LPS/IFN-γ启动成熟后6小时和多至48小时完成遗传工程。在未成熟的DC被工程化改造成过表达免疫抑制分子时,暴露于成熟刺激物后长于24小时的DC的生理学T淋巴细胞抑制活性的重要作用会丧失,关于其原因,我们提出对DC的遗传工程化改造仅与这些DC在遗传工程前(甚至处于前体细胞水平)或之后暴露于成熟刺激物诸如LPS/IFN-γ组合。在不对T淋巴细胞抑制性DC免疫药物进行遗传工程化改造的情况中,此类抑制性DC免疫药物的应用不得不在DC分化的所述抑制窗期间完成,而遗传工程化改造成过表达介导抑制T淋巴细胞活性的分子的T淋巴细胞抑制性DC免疫药物容许对患者的施用有多得多的灵活性。
表1:TLR配体
通过遗传工程化改造在遗传工程前或之后还接受LPS/IFN-γ或类似成熟刺激物的DC以过表达T淋巴细胞刺激分子和阻止免疫刺激窗关闭的分子,DC分化会有可能加宽DC分化的免疫刺激时间窗。它被选择用于通过使用CD40L基因转移来证明本发明的可行性,因为自DC表达的CD40与自活化T淋巴细胞表达的CD40L的相互作用向DC投递有力的活化和成熟信号。优选地,在存在IFN-γ(其在DC成熟中是至关重要的辅因子)的情况中实施此类实验,并且在存在IFN-γ的情况中完成实施例中报告的用CD40L转基因细胞进行的所有实验。可以将与CD40L基因转移相同的原则应用于向DC赋予改善的刺激能力的其它分子。或者,可以如下设计T淋巴细胞抑制性DC免疫药物,即敲除分子诸如CD40或IL-12或类似分子的表达,或自DC免疫药物过表达赋予T淋巴细胞抑制的分子。
通过干扰T-淋巴细胞抑制分子的表达和/或功能,可以使DC分化的免疫抑制窗关闭或者变窄或者移动至较晚的时间点。如下证明此方法的可行性,即敲除干扰DC进行T淋巴细胞活化的分子的表达。实施例部分中显示了T淋巴细胞功能的改善,其通过敲除DC衍生的T淋巴细胞抑制信号(如例如酶IDO,其将活化的T淋巴细胞重度依赖的色氨酸代谢成对活化T淋巴细胞具有促凋亡效果的犬尿氨酸)来实现。作为第二个例子,靶向IL-10(其被认为原型免疫抑制分子,并且它在免疫抑制分化时间窗期间由DC表达)的表达。为了敲除靶分子的表达,优选使用RNA干扰,但是其他技术诸如单链单克隆抗体或反义RNA的胞内表达可以达到相同的目的。或者,所述分子(例如IDO或IL-10)或类似分子的过表达可以用来基于早熟的smDC设计T淋巴细胞抑制性DC免疫药物。实施例部分中的结果(图9)显示了,敲除与IL-10和/或IDO具有相当表达动力学的分子的表达也导致遗传工程化改造的DC的T细胞刺激能力改善。
DC的结构和特性需要以考虑DC发育阶段的动力学方式进行描述。这些中每个阶段可以以某些标志分子的存在与否来表征。这还指明,DC的分子特征依赖于此DC分化的特定阶段和引起DC采用某种分化途径的条件。DC的发育可塑性还解释使用所谓半成熟1型DC(smDC1)(“以释放白介素12表征的T细胞活化性树突细胞”)有利的原因。为了启动从耐受性维持功能向免疫刺激阶段的转换,DC需要被暴露于成熟刺激物(树突细胞成熟剂)。这打开免疫刺激时间窗,在此期间最重要的是,DC释放IL-12作为对向DC制备培养基添加的LPS和IFN-γ或类似试剂的组合的响应。IL-12经由辅助T淋巴细胞上的特定受体起作用,并引起它们呈现Th1表型,从而支持溶细胞性免疫。为了容许此DC/T淋巴细胞相互作用和溶细胞性免疫的形成,优选地,在免疫刺激窗期间的早期时间点时将DC接种入生物体(例如人)中。特别优选的是,在成熟刺激物后6小时注射所述DC。明显地,接种与除去含有树突细胞成熟剂(例如刺激分子LPS和IFN-γ)的DC培养物有关。如下将充分可持续的信号传送入DC中,即2小时,优选4小时,更优选6小时暴露于所述成熟剂(例如LPS和IFN-γ),从而在所述暴露后,DC不可逆地完成成熟过程,而且不再依赖于配体,即DC成熟剂的存在。然而,在形式上,在应用的时候,DC尚没有完成其成熟过程,其花费1-2天。smDC1设计最佳地利用启动成熟后和免疫抑制时间窗打开并开始下调免疫响应前的前24小时期间的免疫刺激时间窗。
在暴露于成熟刺激物诸如LPS/IFN-γ后的早期阶段,DC拥有强烈的免疫活化特性(活化窗,图1),而在其发育的较迟阶段,它们进入免疫抑制阶段(抑制窗,图1)。T细胞活化的分子机制是充分研究和了解的。启动负调节反馈环的事件的分子性质被研究得少得多。如此,依照本发明的DC免疫药物的设计目的在于加宽免疫刺激窗以增强免疫活化,并按比例缩减或关闭免疫抑制窗,如此在DC中阻断负调节反馈环。这是通过如下遗传工程化改造DC实现的,即在遗传工程前或之后将它们暴露于DC成熟剂(诸如LPS/IFN-γ)外过表达免疫刺激基因,或者使用RNA干扰来敲除免疫抑制基因。可以遵循相同的基本原则来调节众多免疫刺激或免疫抑制基因的表达。在实施例部分中显示了此办法的可行性,其通过过表达免疫刺激性CD40L分子或者通过敲除免疫抑制性分子IL-10和IDO来进行。过表达和敲除的组合能增强DC免疫药物的潜力,但是遵循相同的基本逻辑。可以与T淋巴细胞刺激性DC免疫药物类似地设计用于治疗免疫系统的病理学反应过度的T淋巴细胞抑制性DC免疫药物,其如下实现,即遗传工程化改造最初暴露于成熟刺激物(诸如LPS/IFN-γ)的DC,将所得smDC遗传工程化改造成过表达免疫抑制性分子和/或在DC中敲除免疫刺激分子。
依照基于本发明中所描述的遗传工程得到的新型T淋巴细胞刺激性或抑制性DC免疫药物,如下操作部分成熟的smDC,即将编码至少一种免疫刺激性或免疫抑制性分子的核酸分子和/或用以抑制或阻止至少一种免疫抑制性或免疫刺激分子表达的核酸分子,优选核糖核酸分子(例如siRNA)导入所述DC中。
可以通过多种方法来影响或诱导免疫刺激性以及免疫抑制性分子在DC中的表达,由此优选的是,通过导入核酸分子来调控所述表达,如上文所概述的。例如,可以用慢病毒基因转移载体以及脂质体介导的转染实现核酸分子转移。然而,相同的原则在采用其它病毒载体诸如逆转录病毒或腺病毒,或非病毒载体诸如基因枪或聚阳离子技术时或者在采用任何其它基因转移时可适用。
已经开发了数种策略来将外来基因导入细胞中,包括直接注射质粒或DNA脂质体复合物和用经修饰的病毒进行感染。然而,安全和功效是开发使用此类基因转移方法的治疗方案的重要考虑因素。例如,在一种组织的背景中治疗性的蛋白质在另一种中可能是有害的。因而,能限制治疗性序列向合适的细胞进行表达的转录靶向载体是特别想要的。此外,在一些情况中,某些蛋白质可以有治疗窗,从而某些阈值下或上的表达水平分别可以是无效的或毒性的。因此,还会想要创建构建体和设计容许外源控制表达的方法,使得治疗性蛋白质的水平可以随着治疗需要而提高或降低。
可以使用常规的基于病毒和非病毒的基因转移方法来将编码各自分子的核酸或者,备选地,抑制所述分子转录或翻译的核酸(诸如siRNA或反义RNA)导入本发明的DC中。非病毒载体投递系统包括DNA质粒、裸核酸、和与投递载体诸如脂质体复合的核酸。病毒载体投递系统包括DNA和RNA病毒,它们在投递至细胞后具有附加型或整合的基因组。关于基因投递规程的综述,参见Anderson,Science 256:808-813(1992);Nabel和Felgner,TIBTECH 11:211-217(1993);Mitani和Caskey,TIBTECH 11:162-166(1993);Dillon,TIBTECH 11:167-175(1993);Miller,Nature 357:455-460(1992);VanBrunt,Biotechnology 6(10):1149-1154(1988);Vigne,Restorative Neurologyand Neuroscience 8:35-36(1995);Kremer和Perricaudet,British Medical Bulletin51(1):31-44(1995);Haddada等,于Current Topics in Microbiology andImmunology Doerfler and Bohm(编)(1995);及Yu等,Gene Therapy 1:13-26(1994)。
也可以使用小干扰RNA分子。在哺乳动物细胞中,长dsRNA(>30nt)的导入常常启动有力的抗病毒响应,其以对蛋白质合成和RNA降解的非特异性抑制所例示。RNA干扰现象在例如Bass,Nature 411:428-29(2001);Elbahir等,Nature 411:494-98(2001);及Fire等,Nature 391:806-11(1998)中进行描述和讨论,其中还讨论了制备干扰RNA的方法。优选地,本发明中使用的siRNA序列小于100个碱基对,通常为30bp或更短,并且通过本领域中已知的方法来制备。依照本发明的例示性siRNA可以具有多至29bp、25bp、22bp、21bp、20bp、19bp、15bp、10bp、5bp或在其上下或其间的任何整数。
依照本发明的一个优选的实施方案,用于制备未成熟DC的前体获自皮肤、脾、骨髓、胸腺、淋巴结、脐带血或,最优选获自外周血。依照本发明的方法中使用的DC可以直接分离自各自的来源或者衍生自祖细胞。本领域技术人员已知各种方法。例如,可以通过收集抗凝固外周血、造血干细胞,通过白细胞单采血液成分术、或者通过制备暗黄覆盖层、玫瑰花结(resetting)、离心、密度梯度离心(例如使用Ficoll(诸如FICOLLPAQUE)、PERCOLO(用非可透析聚乙烯吡咯烷酮(PVP)包被的胶体二氧化硅颗粒(15-30nm直径)、蔗糖等)、差别溶解细胞、过滤等来分离DC前体和未成熟DC。在某些实施方案中,可以如下制备白细胞群体,诸如例如自受试者收集血液,使其脱纤维蛋白,除去血小板,并使红细胞溶胞。可以使用DC前体、单核细胞、或髓样祖细胞或干细胞来区分iDC。任选地,可以如下自外周血富集单核细胞,即例如利用其附着至塑料表面的能力,经由密度梯度进行离心,单克隆抗体淘选、逆流离心等。若使用通过依照本发明的方法可获得的DC来治疗个体,则iDC可以获自要治疗的个体或者获自与要治疗的个体HLA匹配的健康个体。
可以将DC祖先在合适的培养基中培养和分化。合适的组织培养基包括例如RPMI 1640和DMEM。组织培养基可以补充有人自身或合并的供体血清但不是任何牛来源的血清、氨基酸、维生素、细胞因子诸如GM-CSF和IL-4或IL-13,或IFN-γ,和二价阳离子以促进细胞分化。优选地,也可以将祖细胞在无血清的临床级培养基中培养。与树突细胞培养基一起使用的典型细胞因子组合包含GM-CSF和IL-4或IL-13,或IFN-γ。
为了将成熟刺激物应用于DC,使有效量的至少一种DC成熟剂与iDC接触,所述成熟刺激剂驱动它们进入smDC分化状态(遗传工程前或后或者处于DC前体细胞诸如单核细胞或造血干细胞或前体细胞的状态),其是本发明DC免疫药物的优选状态。优选地,至少一种DC成熟剂选自下组:热灭活的或福尔马林处理的卡介苗(BCG),优选BCG的细胞壁成分、BCG衍生的脂阿拉伯甘露聚糖或BCG成分,自大肠杆菌衍生的脂多糖(LPS)、或灭活的革兰氏阳性或革兰氏阴性微生物、咪唑喹啉化合物,优选咪唑喹啉-4-胺化合物,特别是4-氨基-2-乙氧基甲基-x-二甲基-1H-咪唑[4,5-c]喹啉-1-乙醇或1-(2-甲基丙基)-1H-咪唑[4,5-c]喹啉-4-胺,或其衍生物(参见例如WO00/47719)、合成的双链多核糖核苷酸,优选多聚I:C、天然的双链RNA或RNA病毒或RNA片段、或合成的类似物、或包含未甲基化的CpG基序的合成的或天然的核酸分子。大多数这些化合物是TLR激动剂(参见表1进行比较)。在本发明中,特别优选使用LPS作为树突细胞成熟剂。然而,原则上,单独地或与IFN-γ组合地使用任何TLR激动剂也是可行的。原则上,也有可能将iDC暴露于供成熟用的细胞因子混合物(其通常包括但不限于肿瘤坏死因子α(TNF-α)、IL-1、IL-6、和前列腺素E6)或该组合的部分。此外,有可能触发CD40/CD40L信号传导途径。这可以是如下完成的,即使iDC与处于可溶形式或固定化在表面(例如培养皿或纳米颗粒)上的重组CD40L分子或由CD40L域与另一种蛋白质组成的融合蛋白质诸如IgG-Fc,或者与遗传工程化改造成表达CD40L的原代细胞或细胞系,或者与生理学上上调CD40L表达的活化T淋巴细胞接触。可以与TLR激动剂、炎性细胞因子任意组合地应用CD40/CD40L信号。当然,可以依照本发明使用至少两种所述成熟剂的任意组合。优选地,在存在IFN-γ的情况中使至少一种(优选至少2、3、5、10种)树突细胞成熟剂与树突细胞接触。
依照本发明的另一个优选的实施方案,使遗传工程步骤c)前的iDC与有效量的至少一种树突细胞成熟剂接触至少2小时,优选至少6小时,特别是至少12小时,且最大达24小时。成熟时间取决于多个参数(例如DC成熟剂)。必须选择使得iDC仅部分成熟为smDC的接触时间和其它参数,其使用本领域已知的方法来进行。可以在本领域熟悉的测定法诸如流式细胞术和免疫组织化学中检测细胞表面标志。还可以对细胞监测细胞因子生成(例如通过ELISA,即一种免疫测定法,或通过利用寡核苷酸阵列或蛋白质阵列)。
优选地,能够介导iDC成熟为IL-12释放smDC的至少一种分子选自在存在IFN-γ的情况中的LPS,以便为遗传工程步骤准备好DC以制备具有改善特征的新型T淋巴细胞刺激性或抑制性DC免疫药物。能够使DC能维持其免疫刺激性表型(其特征在于例如IL-12分泌超过约24小时的生理学免疫刺激窗),如此与smDC相比赋予它们卓越的特征的至少一种分子是CD40L,通常但不必需在存在IFN-γ的情况中。本文我们选择使用基于使smDC能够人工表达CD40L(在正常情况下它们并不表达)的办法,其使用上文所概述的遗传工程来进行。然而,不从DC自身而是从辅助原代细胞或细胞系,即活化T淋巴细胞表达CD40L,或者使用可溶性或固定化重组CD40L分子或融合蛋白是可想象的。依照本发明的另一个优选的实施方案,干扰介导T淋巴细胞抑制活性的DC分子表达的至少一种分子选自下组:白介素10(IL-10)和吲哚胺2,3-加双氧酶(IDO)。介导T淋巴细胞抑制活性的分子还可以选自实施例部分的表2和表3中所列的分子,由此在DNA微阵列表达谱分析(profiling)数据方面显示两倍过表达的分子是优选的,但是显示六倍或更高过表达的分子是特别优选的。
依照本发明的一个优选的实施方案,至少一种抗原选自下组:
a)肿瘤抗原、病毒抗原、细菌抗原、或任何其它人微生物或寄生物病原体;或
b)引起变态反应的环境抗原、免疫应答启动所针对的且引起疾病的自身抗原、或移植抗原。
为了生成基于smDC的具有改善特征的新型T淋巴细胞刺激性或抑制性DC免疫药物(其能够针对个体中的抗原诱导特异性增强型免疫应答或增强型免疫抑制),优选地,用至少一种抗原加载iDC,之后使它们与优选的LPS/IFN-γ刺激物接触以制备smDC,接着进行遗传工程。抗原加载对于指示T淋巴细胞针对什么抗原(正是它们必须变为活性的)或者哪种抗原(正是认为它们耐受的)是必需的。供DC装载的抗原可以衍生自患病组织,诸如肿瘤抗原或来自病毒感染细胞的病毒抗原。它们可以是整个死的或活的微生物或者死的或活的原核人或动物细胞,例如人或动物肿瘤细胞或其片段。抗原可以是重组蛋白、或合成肽、编码抗原的基于DNA的病毒或非病毒重组表达载体或者天然的或合成的RNA。或者,抗原可以是已经触发免疫功能障碍诸如变态反应的环境抗原、已经引起疾病的生理性自身免疫应答所针对的自身抗原、或决定器官或干细胞移植排斥的抗原诸如MHC分子。值得注意的是,在供针对异基因移植的耐受性诱导用的T淋巴细胞抑制性DC免疫药物的情况中,加载不可能是必要的,因为器官或干细胞供体DC携带与移植物相同的MHC分子。明显地,在这些后者的情况中,会以如下方式设计DC免疫药物,所述方式使其抑制针对变态反应、移植抗原、或自身抗原的免疫。为了将抗原投递至DC,可以使用多种方法,诸如容许DC吞噬蛋白质或肽抗原的被动暴露,抗原性蛋白质复合物、表达抗原或抗原性肽的细胞或细胞膜、肿瘤细胞胞外体(texosome)、含有抗原或抗原性肽的脂质体、编码抗原或抗原性肽的核酸(可能整合在质粒或病毒载体中)、或来自肿瘤细胞的总RNA。这些方法已经披露于例如W099/03499中。此类载体可以是病毒或非病毒起源的或者可以是纳米颗粒。抗原可以肿瘤抗原、病毒抗原、细菌抗原等,更一般地,寻求的免疫应答或反应所针对的任何肽或多肽。在这方面,可以依照本领域已知的多种技术来使DC对一种或数种抗原致敏。术语“致敏”指明抗原或其部分被暴露在DC(优选与主要组织相容性复合体(MHC)的分子复合)的表面上。原则上,可以在没有用抗原进行在先加载的情况中将DC接种入患者中,并且实现体内摄取抗原,例如通过直接注射入肿瘤中或进入其周围中,进入转移中,或者进入引流淋巴系统(包括淋巴结和一级和/或二级淋巴组织)中。实质上,仅抗原的存在及其向T淋巴细胞的呈递决定DC免疫药物,而不是抗原到达DC的方式。DC加载技术的综述参见RM Steinman和JBanchereau(Nature,卷449/2007年9月27日,第419页-第426页)及其中的参考文献。
可以在多种免疫学功能障碍中使用本发明的抗原加载的且经过遗传改造的DC来治疗性调控免疫应答,这取决于向所述细胞中加载的抗原以及DC所处的生理学功能状态,其通过使用多种信号传导分子诸如DC成熟剂,或通过对DC进行遗传功能化改造来实现。此类功能障碍可以包括但不限于癌症,其可以描述为免疫系统不能排斥经转化的和突变的细胞;传染病,例如在严重和在其它方面不可治疗的微生物感染的语境中或者在免疫受损的个体中,特别在器官或干细胞移植过程中。可以由此类DC免疫药物治疗的其它免疫功能障碍可以源自于免疫学活性过强(例如针对环境抗原),导致变态反应,或者在免疫系统攻击其宿主的情况中,引起自身免疫性疾病。最后,可以基于本发明的方法来设计DC免疫药物,其干扰器官或干细胞/前体细胞移植物(包括通过遗传工程化改造其它细胞所生成的诱导祖细胞(iPS))的排斥,如此促进其宿主对移植物的接受。依照本发明的一个优选的实施方案,至少一种抗原选自下组:肿瘤抗原、病毒抗原、和细菌抗原。可以用个体中应当诱导、抑制、或预防的免疫应答所针对的任何抗原加载依照本发明的经过遗传工程化改造的DC。特别优选肿瘤抗原。
可以如下保藏依照本发明的具有改善的T淋巴细胞刺激性或抑制能力的新型遗传工程化改造DC免疫药物,例如在对患者施用前在成熟为iDC前,部分成熟为smDC后,遗传工程化改造为改善的DC前或后进行深低温保藏。可以使用的深低温保藏剂包括但不限于二甲亚砜(DMSO)、甘油、聚乙烯吡咯烷酮、聚乙二醇、清蛋白、右旋糖苷、蔗糖、乙二醇、i-赤藻糖醇、D-核糖醇、D-甘露醇、D-山梨糖醇、i-肌醇、D-乳糖、氯化胆碱、氨基酸、甲醇、乙酰胺、甘油单乙酸酯和无机盐。
本发明的别的方面涉及一种药物组合物,其包含依照本发明的具有改善的T淋巴细胞刺激或抑制能力的新型遗传工程化改造DC免疫药物。可以使用熟练技术人员公知的方法和组成来将本发明的DC与生理学可接受载体、赋形剂、缓冲剂、和/或稀释剂一起配制。
可以直接对需要免疫调控的受试者施用具有改善的T淋巴细胞刺激或抑制能力的新型遗传工程化改造DC免疫药物。典型地,将约102至约1010个细胞在生理学可接受载体中悬浮。若治疗患有癌症的个体,优选地,将细胞注射入没有疾病的淋巴结中,优选地,进入腹股沟区中,但是任何没有肿瘤或携带肿瘤(转移性)的淋巴结会达到该目的,直接进入肿瘤中或者进入与肿瘤或肿瘤床接近、邻近、或循环或淋巴接触的区域中,或者进入转移性疾病中。可以将DC免疫药物皮下或真皮内应用于皮肤中以容许迁移入淋巴结中。原则上,还有可能将DC免疫药物注射入血流中,作为单一注射或者作为在较长的一段时间里的输注,进入外周血中或者经由导管进入供给患病器官或身体区域的血管(动脉或静脉),或门静脉或肺静脉或动脉等中。可以使用如下的植入释放装置,其将DC药物的连续流投递入肿瘤或转移、淋巴结、血流、或皮肤中。
可以通过适合于施用配方和模式的任何手段来施用本发明的具有改善的T淋巴细胞刺激或抑制能力的新型遗传工程化改造DC免疫药物。例如,可以将细胞与药学可接受载体组合,并用注射器、导管、插管等来施用。如上述的,可以在缓慢释放基质中配制细胞。在以此方式施用时,可以通过适合于所使用的基质的手段来施用配制剂。对本发明可适用的其它施用方法和模式是熟练技术人员公知的。
可以在治疗个体中单独使用本发明的组合物,或者可以与任何其它用于治疗肿瘤的方法组合地使用该组合物。例如,在放射疗法和/或化学疗法(细胞毒性药物、凋亡剂、抗体等);冷冻疗法;近程放射治疗;其它形式的免疫疗法(离体扩充的肿瘤抗原特异性T淋巴细胞、NK细胞、细胞因子和生长因子、肿瘤抗原特异性抗体、或靶向对肿瘤细胞存活至关重要的肿瘤组织的结构诸如血管等);使用病毒或非病毒载体进行的基因疗法等之前、同时、或之后与肿瘤的手术切割术组合使用本发明的方法。此外,本发明的DC免疫药物可以与另一种药剂共施用,所述药剂充当佐剂来使树突细胞成熟和/或加工肿瘤或肿瘤附近或邻近区域内的抗原。还可以以任意组合使用任何和所有这些方法。联合治疗可以是同时的或序贯的,而且可以以任何次序施用,如由治疗医生所确定的。
本发明的另一方面涉及使用依照本发明的树突细胞来制备药物以治疗和/或预防癌症和/或微生物或寄生物感染;或者以治疗和/或预防变态反应、自身免疫性疾病、或干细胞或器官移植排斥。优选地,可以在癌症预防和/或癌症治疗中采用依照本发明的部分成熟的树突细胞。在此类情况中,用至少一种肿瘤抗原加载树突细胞。例如,但不限于,可以将细胞直接施用入肿瘤中,手术除去或切除肿瘤后以肿瘤旁进入肿瘤床中,进入与肿瘤直接接触的引流淋巴结中,进入引起或喂养肿瘤或受肿瘤所累的器官的血管或淋巴导管(例如门静脉或肺静脉或动脉等)中。
可以在肿瘤的其它治疗诸如化学疗法或放射疗法同时或之后应用本发明的部分成熟的树突细胞的施用。此外,本发明的部分成熟的树突细胞可以与另一种药剂共施用,所述药剂充当佐剂来使树突细胞成熟和/或加工肿瘤或肿瘤附近或邻近区域内的抗原。还可以以任意组合使用任何和所有这些方法。联合治疗可以是同时的或序贯的,而且可以以任何次序施用,如由治疗医生所确定的。另外,还可以将树突细胞配制或混合入缓慢释放基质中以植入肿瘤或肿瘤床中或周围的区域中,使得细胞被缓慢释放入肿瘤或肿瘤床中,以便与肿瘤抗原接触。
依照本发明的一个优选的实施方案,在放射疗法和/或抗肿瘤或抗微生物化学疗法、或目的在于治疗变态反应、自身免疫性疾病、或干细胞或器官移植排斥的任何疗法前、同时、或之后对个体施用所述药物。可以与其它癌症疗法组合采用依照本发明的树突细胞以便实现甚至更有益的效果。
本发明的另一方面涉及依照本发明的树突细胞用于制备药物的用途,所述药物用于治疗和/或预防由免疫系统针对环境抗原(诸如变态反应),或针对自身免疫性疾病过程中的自身抗原的生理性反应过度引起的免疫学疾病。
优选地,在目的在于治疗或预防变态反应或自身免疫性疾病的其它药征前、同时、或之后对个体施用所述药物。
本发明的别的方面涉及依照本发明的树突细胞用于制备药物的用途,所述药物用于治疗和/或预防异基因干细胞移植的免疫学排斥(优选地,在治疗血液学恶性肿瘤中使用),或者用于治疗和/或预防异基因器官移植的排斥。
优选地,在目的在于治疗或预防异基因干细胞或器官移植排斥的其它药征(modality)前、同时、或之后对个体施用所述药物。
本发明通过以下各图和实施例进一步例示,然而,不限于此。
图1在DC分化过程的动力学图示中显示了DC的发育可塑性。
图2显示了smDC1基础设计的质量对照。
图3显示了CD40L基因转移的结果。
图4显示了IL-12和IL-10分泌的数量和质量。
图5显示了溶细胞活性的潜力(正方形,CD40L转基因DC;菱形,GFP转基因DC;三角形,对照DC)。
图6显示了敲除IL-10表达的LPS活化DC的免疫刺激能力。
图7显示了具有沉默的IDO表达的LPS活化DC的免疫刺激能力。
图8显示了DC表达谱分析实验的实验设计。
图9a显示了使用RNA干扰敲除DC中在表达谱分析实验中鉴定的靶分子的表达后的增殖响应改善的例子。图9b显示了如下基因的别的例子,所述基因在用siRNA敲除其在DC中的表达后导致此类经遗传工程化改造的DC对异基因淋巴细胞的刺激能力改善,如与对照siRNA转染或未转染的DC相比所指明的,如所标示的。
实施例:通过遗传工程制备T淋巴细胞刺激性或抑制性DC免疫药物的方法。
白细胞单采血液成分术
使用Amicus白细胞单采血液成分术装置(Baxter,Deerfield,IL)从健康志愿者和在得到负责的机构审查委员会批准的临床试验背景中治疗的患有各种瘤形成的患者收集白细胞。依照世界医学协会(World Medical Association)赫尔辛基宣言,所有个体均对这些研究给予他们的知情同意。在Sysmex细胞计数器(Sysmex,Bornbarch,德国)上和/或通过流式细胞术测定细胞数目和子集。
单核细胞富集
使用补充有1%人合并AB血浆(Octaplas,Octapharma,Vienna,奥地利)的AIM-V(Invitrogen,Carlsbad,CA)或CellGro培养基(CellGenix,Freiburg,德国)通过塑料粘着来富集单核细胞,如先前所描述的。对于线内规程,我们遵循制造商提供的指令。使用Elutra细胞分离器(Gambro BCT,Lakewood,CO),通过在维持2400rpm的离心速度时加载入淘析室中来从白细胞单采血液成分术产物富集单核细胞。此后,离心速度和淘析培养基(PBS/HSA Baxter,NewJersey,NJ)流动保持不变以进行细胞分级。或者,用CliniMACS细胞选择系统(Miltenyi,Bergisch Gladbach,德国)完成对单核细胞的选择,所述CliniMACS细胞选择系统使用经CD14包被的磁珠在磁性柱中保留单核细胞。用于单核细胞富集的另一种选项是使用Isolex 300i磁性细胞选择器(Nexell,Irvine,CA)来完成T和B淋巴细胞的消减以富集单核细胞。如下在磁性柱中保留淋巴细胞,即使它们与经CD2和CD19包被的磁珠接触,并收集流过液。通过流式细胞术表征所有富集规程的最终产物。
流式细胞术
使用Trucount系统(Becton Dickinson,New Jersey,NJ)分别通过用抗CD45-FITC、抗CD3-PerCP、抗CD19-APC、抗CD14-APC、和抗CD15-FITC(BD Pharmingen San Diego,CA)标记的抗体对白细胞单采血液成分术和单核细胞富集产物分析总体淋巴细胞、T淋巴细胞、B淋巴细胞、单核细胞、和粒细胞。在FACSCalibur流式细胞仪(Becton Dickinson,Mountain View,CA)上分析经标记的细胞。合适的同种型对照抗体包括在分析中。
DC制备
以1x106个单核细胞/cm2的密度在补充有2%合并人AB血浆的AIM-V培养基中或者在CellGro培养基中于37℃在湿润培养箱中培养通过上文所述的各种富集规程分离的单核细胞达6天。培养基补充有1000U/ml人GM-CSF和300U/ml人IL-4(均来自CellGenix,Freiburg,德国),并在第3天用相同体积的AIM-V/2%OP或CellGro加上GM-CSF和IL-4替换。在第6天时如下实施成熟,即将50ng/ml IFN-γ(Boehringer Ingelheim,Vienna,奥地利)和脂多糖(LPS,大肠杆菌菌株O111:B4,Calbiochem,San Diego,CA,USA)(范围为1-1000ng/ml)添加至培养物达6小时以生成半成熟(sm)DC,随后将其冷冻;用临床级LPS(US药典,Bethesda,MD)制备患者的DC疫苗。
DC免疫表型分析
使用下列抗体来测定DC的成熟状态:抗CD86-APC(BD Pharmingen,SanDiego,CA)、抗CD80-PE(Immunotech,Beckman Coulter,Fullerton,CA)、抗CD83-APC(三种都来自BD Pharmingen,San Diego,CA)、抗MHC I-PE、抗MHC II-FITC(两者都来自Dako Cytomation,Carpinteria,CA)、和抗CD45-PerCP(BD Pharmingen,San Diego,CA)。通过碘化丙啶染色(Sigma,St.Louis,MO)来测量DC的成活力。使用FACS Calibur流式细胞仪来分析细胞。合适的同种型对照抗体包括在分析中。
通过ELISA进行的IL-12检测
如先前所描述的那样测量DC培养物的上清液中的IL-12浓度。
异基因混合的白细胞反应
通过梯度离心自外周血分离异基因应答PBMC。一式三份将刺激性DC(10000个、2000个、或400个)与105个应答细胞在96孔圆底平板上在200μl补充有2%合并人血浆的AIM-V培养基中放置在一起。对于阳性参照,用100ng/ml葡萄球菌肠毒素A/B(SEA/SEB,Toxin Technologies Inc.,Sarasota,FL)刺激105个应答细胞。在第4天,将共培养物与1μCi氚胸苷溶液(NEN LifeScience Products,Boston,MA)一起再温育18小时。最后,用Skatron(Lier,Norway)收集器收获细胞。使用Trilux读板仪(Wallac Oy,Turku,芬兰)来计算掺入的氚胸苷。或者,用CFSE(Molecular Probes,Eugene,OR)标记异基因PBMC,并以1/5、1/10、1/20、1/40、和1/80的比率与DC混合。对于对照,没有添加DC或SEA/SEB。最后,用抗CD3-PerCP标记PBMC,并使用FACSCalibur流式细胞仪来分析。测定CD3阳性CFSE阴性T淋巴细胞的百分比。
向smDC中进行慢病毒基因转移
使用ViraPowerTM慢病毒表达系统(来自Invitrogen),通过与编码病毒结构蛋白、聚合酶和逆转录酶的pLP质粒(pLP/VSVG、pLP-1、pLP-2)和含有GFP或CD40L的质粒共转染293FT生产细胞系来生成慢病毒颗粒。共转染后72小时,收获所有上清液,并通过超离心浓缩100x。在上文所概述的条件下培养DC,并使其成熟。分别在启动成熟后48小时或6小时收获DC。然后用与6μg/mlPolybrene(来自Sigma-Aldrich)加上标准浓度的IL-4、GM-CSF、和IFN-γ组合的慢病毒颗粒(250μl 100x浓缩的慢病毒上清液/1x106个DC)转导早熟的smDC。对于IL-12质量对照,在24小时后采集上清液,并在48小时后遵循标准规程来测量GFP/CD40L的表达。
DC中的RNA干扰
依照上文所概述的标准规程来制备DC。在第6天,使用转染试剂(Dharmacon)依照制造商的指令用100pmol基因特异性siRNA转染106个DC。转染后12小时,用LPS/IFN-γ刺激DC达6小时。所有应用均与上文所概述的方法类似。
结果
目前使用中的DC免疫药物采用单核细胞衍生的DC,其加载有任何性质的抗原,如介绍中所概述的,并暴露于具有触发IL-12从DC释放的能力的成熟刺激物。以IL-12分泌表征的DC表型具有诱导支持溶细胞免疫的1型免疫系统极化的能力。这暗示刺激性DC免疫药物需要在IL-12分泌的时间窗期间应用于患者以容许抗原在存在IL-12的情况中从DC呈递至T淋巴细胞(图1)。具有改善的T淋巴细胞刺激或抑制能力的新型遗传工程化改造DC免疫药物克服此限制,因为对DC的遗传操作容许免疫刺激窗保持开放达较长的时间期限。
在以下实施例中,已经制备和研究经遗传工程化改造的DC免疫药物。使用慢病毒基因转基因或基于脂质体的转染来将DNA或RNA投递入DC中,但是可以假设,任何核酸投递技术可以在所述能力中起作用。作为免疫刺激基因在DC免疫药物中过表达的例子,使用慢病毒基因转移来工程化改造DC以表达CD40L分子。功能研究确认,此类工程化改造DC免疫药物具有增强的刺激免疫应答的潜力。此外,证明了敲除免疫抑制性分子IL-10和IDO也增强刺激能力,其通过用设计用于RNA干扰IL-10和IDO的siRNA分子工程化改造DC来实现。为了鉴定牵涉免疫抑制反馈环的别的DC分子,使用DNA微阵列进行全基因组DNA表达谱分析。基于为与IL-10或IDO的基因表达谱具有类似表达谱的基因分组的簇分析,发现基因列表具有负调节免疫应答的潜力。如此,在DC免疫药物中敲除这些基因会改善其免疫刺激能力。因此,为了容许对规定的免疫系统成分进行特定调控而遗传工程化改造的DC免疫药物实现相关免疫系统功能障碍的治疗。最后,显示了使用RNA干扰敲除DC表达谱分析实验中以与IL-10或IDO类似的动力学表达的靶基因的功能后果的例子。在与异基因T淋巴细胞的共培养物中,观察到此类经遗传工程化改造的DC触发增殖的能力改善,指明增强的T淋巴细胞刺激。
图2显示了用于遗传工程的smDC1基础设计的质量对照。DC免疫药物必须满足规定的质量对照标准。小图A显示了自外周血单核细胞制备的DC的纯度、成活力、和产量。通过白细胞单采血液成分术来收集此类单核细胞,并通过逆流离心(淘析)来富集单核细胞。在存在IL-4和GM-CSF的情况中,单核细胞在6天内在体外分化成iDC。用抗原加载iDC,随后暴露于由LPS和IFN-γ组成的成熟刺激物达6小时,并将其冷冻。在此阶段。它们称作半成熟的,因为虽然它们不可逆地继续其成熟,但是它们尚未显示mDC的典型表型和功能特征。最重要地,在所述阶段(免疫刺激窗,启动成熟后约0-24小时,图1),DC触发成熟,反之在较晚的阶段(免疫抑制窗,启动成熟后约24-48小时,图1)。然而,在临床应用中,在此分化阶段将它们注射入患者中,在那里它们完成其成熟,并触发免疫应答(24小时前),但是随后(在约24小时时)也进入-依照其由成熟刺激物触发的生理学发育程序-免疫抑制阶段(我们旨在通过遗传工程化改造DC免疫药物来阻止)。
对于质量对照,将一等分试样的DC免疫药物融化,并再培养2天以便让DC完成其成熟过程。这两天期间,它们分泌细胞因子,最重要的是IL-12(成熟后早期)和IL-10(成熟后晚期)(小图B;显示了来自三个个体的均值±SEM)。还有,它们显示了至关重要的DC膜分子的表达样式的变化(小图C)。最后,通过以指定的比率与经CFSE标记的异基因PBMC共培养(其触发与CFSE的稀释度和荧光降低有关的细胞分裂)来对DC进行alloMLR潜力测试(小图D)。柱形图显示了来自三个个体的增殖细胞的均值±SEM百分比。
初始刺激物对于启动免疫抑制反馈环也是必要的。一般而言,响应特定刺激物的活化越强,反馈信号传导会越强,以便将免疫活化下调至其基线水平,由此阻止免疫应答失去控制和引起自身免疫性疾病。如此,发现成熟刺激物LPS/IFN-γ导致最高量的IL-12释放,而且导致最高量的IL-10释放。
表2:供癌症接种用的smDC1的基础设计的规格
测试 | 规格 | 测试 | 规格 |
纯度 | 70-100% | IL-12 | >100pg/ml |
成活力 | 70-100% | alloMLR | |
表型 | DC∶MNC=1∶5 | >30% | |
CD80 | 60-100% | DC∶MNC=1∶10 | >30% |
CD86 | 60-100% | DC∶MNC=1∶20 | >15% |
MHC I | 60-100% | 阴性对照 | <10% |
MHC II | 60-100% | BACTEC | NEG |
CD83 | 60-100% | 支原体 | NEG |
CD14 | 0-40% | HIV 1/HIV 2 | NEG |
IgG对照 | <1% | HBV/HCV | NEG |
经由过表达CD40L分子增强的刺激性DC免疫药物的描述。
为了加宽以分泌IL-12表征的DC的免疫刺激窗(图1),将DC遗传工程化改造成过表达CD40L。此分子通常自活化T淋巴细胞表达,并与DC上的CD40相互作用,将至关重要的活化信号传送入DC中。将此实验设计为将活化分子转移入DC免疫药物中的一个例子。然而,原则上,可以对其它刺激分子使用相同的规程或者,为了设计抑制性DC免疫药物,可以自DC过表达免疫抑制性分子。具体地,CD40L基因转移入DC中的基本原理是
(i)容许DC变为与活化T淋巴细胞无关以将CD40L信号投递至DC;
(ii)在本实验中假设并发现,由于在DC自身上通过从组成性活性启动子的表达实现CD40L的连续存在,使DC能够分泌IL-12达如下的时间期限,其比在DC受到常规成熟刺激物诸如LPS/IFN-γ时长得多;
(iii)与单独的LPS/IFN-γ或CD40L/IFN-γ刺激物相比自DC分泌的IL-12总量是高得相当多,并且IL-12分泌的动力学在性质上已经有所不同,在应用刺激物后更早开始,如此加宽DC分化的免疫刺激窗;
(iv)暴露于LPS/IFN-γ后甚至48小时,此时DC已经耗尽其分泌IL-12的能力,CD40L基因转移使DC能够开始IL-12分泌的第二阶段(图3)。
图3显示了CD40L基因转移。在小图A中,显示了慢病毒基因转移后在6小时smDC和48小时mDC中的GFP或CD40L表达。将早熟的DC暴露于慢病毒载体后48小时完成本文显示的所有实验。与iDC、暴露于单独的LPS/IFN-γ的DC、或经GFP工程化改造的6小时smDC和48小时mDC相比自DC表达CD40L引起IL-12的分泌增强(小图B)。GFP基因转移后增强的IL-12释放可能是由病毒双链RNA引起的,所述病毒双链RNA经由6小时smDC中但不再在48小时LPS/IFN-γmDC中表达的TLR(参见表1)发信号。通过向DC(黑色直方图,未成熟的DC;白色直方图,mDC)中进行慢病毒基因转移没有改变功能上重要的DC膜分子的表达谱(小图C)。
细胞因子IL-12和IL-10的分泌在暴露于LPS/IFN-γ、CD40L/IFN-γ、或两者的组合的DC中在质量和数量方面有所不同(图4)。与单独的CD40L/IFN-γ刺激物相比,IL-12的分泌在应用组合刺激物时高几乎两倍,而且与单独的LPS/IFN-γ刺激物相比也是高得相当多的。另外,来自暴露于LPS/IFN-γ/CD40L组合刺激物的DC的IL-12分泌在12小时后已经明显地以相当大的量可检出,而LPS/IFN-γ和CD40L/IFN-γ仅在暴露于初始成熟信号后12和24小时之间触发生物学相关水平的IL-12分泌。这种观察与加宽DC分化的免疫刺激窗以便改善DC免疫药物的刺激能力的本发明目的一致。IL-10的最大限度表达在DC暴露于LPS/IFN-γ/CD40L或单独的CD40L/IFN-γ时是相似的,但是在仅对DC成熟使用LPS/IFN-γ时较低。然而,免疫抑制性细胞因子IL-10在CD40L/IFN-γ信号传导后12小时后已经以生物学相关水平可检测,而组合刺激物LPS/IFN-γ/CD40L显示与那些仅LPS/IFN-γ成熟的DC的动力学类似的动力学。CD40L/IFN-γ刺激后的IL-10早期释放负干扰DC分化的免疫刺激窗,并且应当(在设计免疫刺激性DC药物的情况中)避免。推断与应用单独的LPS/IFN-γ或CD40L/IFN-γ相比,组合刺激物LPS/IFN-γ/CD40L的免疫刺激能力与免疫抑制能力间平衡的净效果(考虑IL-12和IL-10的分泌样式)明显朝向改善的免疫刺激能力。与较早的出版物形成对照,本文所使用的DC会接受成熟刺激物的组合。在生理学上,DC能活化T淋巴细胞的阶段(以IL-12分泌表征)和DC会抑制T淋巴细胞活性的第二阶段(以IL-10分泌和由酶IDO活性引起的色氨酸消减表征)会通过使iDC与足够的成熟刺激物诸如LPS/IFN-γ接触来触发(图1)。本文证明了初始暴露于LPS/IFN-γ(或者在存在IFN-γ的情况中的另一种TLR激动剂),接着将DC遗传工程化改造成过表达诸如CD40L等分子维持能够T淋巴细胞活化的DC表型,并阻止DC呈现抑制表型(图4)。IL-12的分泌在存在IFN-γ的情况中经由TLR信号传导途径实现的初始成熟后6小时或48小时完成遗传工程时维持得长于20-24小时的生理学时间窗。
图4显示了IL-12和IL-10分泌的数量和质量。在存在IFN-γ的情况中将DC暴露于指定的成熟刺激物。在指定的时间点测量IL-12和IL-10在培养物上清液中的浓度。
在遗传工程化改造T淋巴细胞刺激性DC免疫药物的本设计中特别重要的是IL-12分泌DC具有经由1型免疫应答极化触发溶细胞免疫的能力。如此,通过分析粒酶B在CD8阳性CTL中的含量来进一步调查暴露于CD40L转基因DC的T淋巴细胞触发溶细胞免疫应答的潜力(图5)。确实,发现与对照GFP转基因DC或未转导mDC相比,CTL与CD40L转基因DC的共培养导致粒酶B表达明显增强。这是此类CTL的溶细胞潜力改善的一项强烈指标,如此提供来自DC免疫药物的CD40L表达具有改善的免疫刺激能力的证据。
图5显示了溶细胞活性的潜力。与GFP转基因DC(菱形)和未转导的mDC(三角形)相比,CTL的总百分比在将PBMC与CD40L转基因DC(左侧小图,正方形)共培养时仅略微升高。在分析与CD40L转基因DC共培养的CTL中的粒酶B表达时,与GFP转基因DC(菱形)和未转导mDC(三角形)相比,发现明显升高(右侧小图,正方形)。
通过敲除免疫抑制性细胞因子IL-10的表达的工程化改造得到的具有改善的T淋巴细胞刺激能力的DC免疫药物的描述
基于其中敲除介导免疫抑制的分子表达的DC免疫药物的假设,设计实验来阻断DC中的IL-10基因表达,其通过使用4种靶物特异性siRNA的集合进行RNA干扰来实现(图6)。这导致LPS/IFN-γ活化DC中非常一致且可再现的IL-10表达敲除,与对照沉默的mDC相比导致较高的IL-12分泌。此观察暗示自分泌途径,其基于自DC分泌的IL-10结合同一DC上的IL-10受体,导致下调的IL-12生成。不同于发现经遗传工程化改造的与正常的DC之间没有免疫表型差异,如通过CD80、CD86、MHC I类、和II类表达所评估的。最重要的是,在alloMLR中,观察到与对照实验相比,为了抑制IL-10分泌以活化T淋巴细胞而工程化改造的DC免疫药物的大得相当多的潜力。
另外,CD25+FoxP3+细胞在CD4+T细胞群体(推测为抑制免疫应答的调节性T细胞(Tregs)群体)中的百分比降低,可能是由于LPS/IFN-γ活化的DC中的IL-10沉默。
图6显示了通过遗传工程阻断IL-10表达的LPS/IFN-γ活化DC的免疫刺激能力。用LPS/IFN-γ活化前12小时,用4种IL-10特异性siRNA的集合或非特异性对照siRNA转染DC。然后用6小时LPS成熟的DC(mDC)(或是IL-10(黑色柱形)或是对照沉默的(白色柱形))以1∶3=DC∶T细胞比率刺激分离的异基因CD3+T细胞。使用Trucount系统和FACS LSRII流式细胞仪在共培养的第6天分析CD4+、CD8+、(小图A)和CD4+CD25+FoxP3+T细胞(小图B)。LPS/IFN-γ活化后48小时分别通过流式细胞仪和ELISA来测量免疫表型以及IL-10和IL-12分泌(小图C)。免疫表型分析在IL-10沉默的DC(小图D)或对照沉默的DC(小图E)中比较LPS/IFN-γ活化的DC(白色直方图)与iDC(黑色直方图)。
为了敲除免疫抑制性酶IDO的表达而工程化改造的T淋巴细胞刺激性DC免疫药物的描述
使用siRNA来敲除已知的免疫抑制性效应分子IDO的表达(图7)。为了优化siRNA转染和IDO敲除的效率,首先使用用IFN-γ活化的HeLa细胞。随后,在经优化的条件下转染DC。在HeLa细胞和DC两者中,可以沉默IDO的表达,如Western印迹实验中所表明的。在alloMLR潜力测定法中使用IDO沉默的DC、用杂混的对照siRNA转染的DC、和iDC作为刺激物。观察到与用混杂的siRNA转染的DC或iDC相比IDO沉默的DC的刺激潜力是大得相当多的。这适用于CD8+CTL以及CD4+Th细胞。
图7显示了具有沉默的IDO表达的LPS/IFN-γ活化DC的免疫刺激能力。首先,使用Western印迹实验来证明在HeLa细胞(小图A)以及DC(小图B)中有效的IDO敲除。为了调查IDO沉默的DC对CD8+CTL(小图C)和CD4+Th淋巴纽胞(小图D)的刺激潜力,将PBMC与IDO沉默的DC(正方形)、对照沉默的DC(菱形,scra=序列混杂的)、或iDC(三角形)共培养。在所有情况中,IDO沉默的DC的刺激能力优于对照。
免疫抑制分子
使用全基因组DNA微阵列来产生暴露于成熟刺激物LPS/IFN-γ、CD40L/IFN-γ信号传导、或LPS/IFN-γ/CD40L信号传导的组合,以及合适的对照的DC的表达谱(图8)。
图8显示了DC表达谱分析。将DC暴露于指定的成熟刺激物或者维持未成熟。在指定的时间点提取RNA,并使用全基因组DNA微阵列来进行表达谱分析。使用CarmaWeb(基于全面R的微阵列分析,Bioinformatics Graz and theTyrolean Cancer Research Institute,奥地利)来分析表达谱分析的结果。将所有数据分成20个簇,其使用CarmaWeb软件平台的基本算法,并鉴定含有IDO和IL-10的簇。在这些簇中的基因与两种已知免疫抑制性DC分子具有类似的表达谱,这得出如下结论,即它们在DC的免疫调节中具有也是免疫抑制性的功能(表3和表4)。
表3:IDO表达簇。依照CarmaWeb算法,所列基因具有的表达谱类似于IDO(基因名称INDO)的表达谱,提示与IDO功能类似的功能(数目是相对于未成熟DC的log,其中底数为2)。
6小时 LPS 12小时 LPS 24小时 LPS 48小时 LPS
IFN-γ对 6小 CD40L IFN-γ CD40L IFN-γ CD40L IFN-γ
独特的ID 名称 时iDC M 对12小时iDC M 对24小时iDC M 对48小对iDC M
210118_s_at IL1A 12,5712385 12,656363 11,625489 9,828246
1405_i_at CCL5 9,775104 10,949544 12,230392 11,143438
1552995_at IL27 10,449822 10,585213 10,862312 8,519849
1554997_a_a
t PTGS2 11,7640085 9,820589 11,614364 11,624819
1555759_a_a
t CCL5 12,916491 12,965171 12,63708 12,723415
1556378_a_a
t LOC401530 5,897853 10,2600975 12,640888 13,004906
1570388_a_a
t LOC401530 3,8191023 9,072843 11,445329 11,711242
202269_x_at GBP1 8,517265 8,2832775 8,787927 8,968534
202411_at IFI27 9,403024 9,560578 9,054876 8,233445
203828_s_at IL32 9,310799 8,968001 10,09564 9,038413
203915_at CXCL9 13,417887 13,097824 12,633152 11,7289915
204439_at IFI44L 11,743961 9,8876705 7,0858197 6,4474745
204470_at CXCL1 10,931133 8,087279 8,933327 5,9426484
204533_at CXCL10 9,392692 10,44239 10,786983 11,601183
204655_at CCL5 9,542912 11,558153 11,215681 11,169104
204698_at ISG20 12,59375 10,52378 9,431844 8,965937
204748_at PTGS2 12,489249 7,144587 7,9113717 8,436964
205013_s_at ADORA2A 10,245063 8,496423 10,054197 9,867236
205067_at IL1B 12,89035 7,7892747 11,259029 11,700619
205207_at IL6 7,463433 7,6129217 8,461436 8,870339
205476_at CCL20 12,470199 12,587071 13,649405 12,875935
205569_at LAMP3 6,9127846 9,671546 9,207934 10,519818
205599_at TRAF1 8,448494 9,332896 9,679705 11,050019
205680_at MMP10 6,9289026 9,5307 11,131636 11,908984
205681_at BCL2A1 12,63274 12,276044 12,321126 12,00514
205692_s_at CD38 11,776373 10,918448 8,890723 6,572375
205890_s_at UBD 11,738066 11,718061 12,080868 11,735866
206025_s_at TNFAIP6 12,46147 9,442592 10,791083 10,010692
206026_s_at TNFAIP6 8,55364 8,492605 8,934455 8,366126
206337_at CCR7 8,625213 10,63397 11,733897 11,844164
206341_at IL2RA 7,962748 9,035077 10,360973 8,479506
206765_at KCNJ2 4,7675686 9,681816 10,2331705 9,70106
206881_s_at LILRA3 9,8311825 9,066225 9,314402 5,396609
207113_s_at TNF 12,704884 13,268166 11,278352 8,395802
207160_at IL12A 9,43968 13,149242 13,380418 8,626079
207176_s_at CD80 8,31917 7,76368 8,680011 8,573913
207375_s_at IL15RA 9,771918 10,090428 10,584916 10,606205
207536_s_at TNFRSF9 6,231527 7,7779512 9,701627 9,752483
207901_at IL12B 12,714132 13,101075 13,353125 7,458026
209813_x_at TRGV9 3,9887655 10,498012 10,522085 8,133455
210029_at INDO 12,789924 10,134047 12,052269 12,43933
210072_at CCL19 7,2848625 10,570788 12,461668 11,943225
210163_at CXCL11 13,109848 12,912714 11,681597 10,053444
210511_s_at INHBA 12,3267975 12,957313 10,577578 10,25125
211122_s_at CXCL11 13,135339 13,034375 12,199861 9,8954315
211269_s_at IL2RA 7,485799 9,023119 10,643187 10,435802
213497_at ABTB2 6,1966333 8,42709 9,347927 9,879564
215806_x_at TRGC2 4,249278 10,289696 10,347067 7,9237046
217546_at MT1M 6,80711 8,103879 9,402974 8,6524935
219159_s_at SLAMF7 12,195147 10,547217 8,994919 6,4161515
219424_at EBI3 10,374818 11,288197 12,070472 10,607756
220054_at IL23A 6,8979635 12,20336 13,634168 11,491903
222838_at SLAMF7 12,751745 12,511322 10,977879 8,82794
226560_at SGPP2 10,281151 9,231938 9,82061 8,582578
227140_at INHBA 12,689469 10,28581 10,5587015 10,436948
227180_at ELOVL7 8,1362 8,21396 9,105779 9,419119
229437_at BIC 9,607157 11,00861 11,168569 11,648725
229625_at GBP5 11,195785 11,012673 7,085479 8,598587
231577_s_at GBP1 7,464914 8,356869 8,556055 8,40867
235229_at 8,6328745 8,228744 8,894646 8,620824
238439_at ANKRD22 7,496807 7,9435267 8,688104 10,303036
238581_at GBP5 9,960986 9,9203 7,430702 7,571562
238725_at 5,625349 7,5093164 9,704128 10,179434
240287_at LOC341720 10,947632 11,357612 9,703499 4,2557507
242814_at SERPINB9 9,147152 9,357069 9,150615 9,138746
33304_at ISG20 11,309338 7,729137 8,4003 6,2511134
39402_at IL1B 12,057478 6,510902 10,422022 11,546757
228439_at MGC20410 6,443625 4,5576925 5,1437 2,8559468
232078_at PVRL2 6,135475 4,746409 3,8879604 4,522022
1561908_a_a
t HS3ST3B1 7,0928926 4,458722 3,670319 3,8414023
204141_at TUBB2 5,3551335 4,7444496 4,0537806 5,1056914
207275_s_at ACSL1 5,8632264 5,869882 4,1483216 4,095168
210563_x_at CFLAR 5,100706 5,0670333 5,419278 4,1996965
210564_x_at CFLAR 5,380331 4,9984617 5,3778234 4,160804
218400_at OAS3 6,8762517 6,1737237 4,410526 2,8219512
220132_s_at CLEC2D 4,4514236 6,669927 5,78131 1,7744006
222303_at ETS2 4,60927 5,654717 5,4186363 3,4308143
229221_at CD44 7,1867433 5,771355 3,1319969 4,7465234
230499_at BIRC3 4,71687 4,9918036 5,3390183 5,366412
232682_at DSU 4,6348085 5,7252564 5,5562453 3,688244
243296_at PBEF1 6,237873 4,170852 4,781295 5,1768007
243894_at SLC41A2 7,1203766 4,7231464 4,238267 4,4368286
1554539_a_a
t RHOF 5,002441 4,1752563 5,4232445 5,0010266
1563357_at SERPINB9 6,368476 5,3010783 3,9647322 5,4146433
202509s_at TNFAIP2 5,195548 5,9835477 4,9414105 3,823941
203287_at LAD1 4,5966916 6,1679506 5,8620677 3,8349202
204715_at PANX1 6,0651593 4,6488233 5,01669 4,461601
204794_at DUSP2 5,4158106 3,7610755 4,858317 6,9610953
207389_at GP1BA 6,946879 6,472854 5,3776803 2,2710993
209039_x_at EHD1 5,992218 4,9087305 4,788946 5,520831
209928_s_at MSC 4,9872894 5,956739 4,365724 5,714541
215078_at SOD2 6,952513 7,3351035 3,1302252 3,7524989
216336_x_at MT1M 4,864737 4,9637637 6,200034 5,143623
219716_at APOL6 7,687471 5,4070616 4,6342998 3,8593137
221779_at MICAL-L1 5,128767 5,6324835 6,08239 4,3245826
226189_at 7,0649176 5,7119026 5,3064666 3,6578546
227014_at LOC57168 7,947885 4,03786 4,678014 4,4947524
232304_at PELI1 6,7769756 5,062101 4,4606757 5,45498
234985_at LOC143458 5,5271115 4,7101035 5,720184 4,8197145
242649_x_at C15orf21 6,9237723 5,3964243 4,532885 4,963293
1559391_s_a
t B4GALT5 4,598228 7,0806437 5,126474 4,786352
200629_at WARS 6,951068 5,0352116 4,546803 4,863137
202688_at TNFSF10 6,8965373 5,708206 4,570576 5,1128407
202748_at GBP2 5,793167 6,4275317 4,901077 4,8227687
203685_at BCL2 7,285017 5,9438853 4,1533656 4,8756795
204015_s_at DUSP4 8,879749 6,673204 3,3034465 4,2480526
204926_at INHBA 5,6395097 7,8953505 4,531301 3,1433787
206157_at PTX3 7,3603053 5,5506916 5,1639824 4,220827
209803_s_at PHLDA2 5,2155585 4,638463 6,690672 5,015275
209939_x_at CFLAR 5,3425546 5,3650227 6,3261905 4,451903
211302_s_at PDE4B 9,388175 4,2461367 4,496878 4,7590027
215671_at PDE4B 6,8900924 4,9770446 4,6578245 5,777932
216705_s_at ADA 6,1992345 4,8220434 5,4637165 5,23145
218943_s_at DDX58 7,6221895 6,2795057 4,79867 4,15005
219014_at PLAC8 4,8591843 4,901381 8,194876 3,2458937
221087_s_at APOL3 6,84244 5,7354436 4,5675263 5,530723
221185_s_at IQCG 6,629468 3,8091857 6,4129505 5,9112988
222812_s_at RHOF 5,2598224 5,562695 5,265753 5,8453097
239876_at NFKB1 6,668459 5,936471 4,785418 5,0308824
240013_at 5,927128 5,32223 5,1699376 5,818874
242234_at BIRC4BP 5,92746 5,946681 4,6361747 6,306454
35150_at CD40 5,5139947 5,5919642 6,1309977 5,227399
1553713_a_a
t RHEBL1 5,2900615 5,984809 5,9541264 5,9824634
1570253_a_a
t RHEBL1 5,2768035 6,012619 5,687341 5,871174
202687_s_at TNFSF10 7,8631916 5,812222 4,413325 5,5924144
204415_at G1P3 4,8981137 6,400349 6,9897156 3,8297942
205483_s_at G1P2 6,6436443 6,099025 5,6568613 4,5633087
206975_at LTA 7,6997614 8,081627 5,2525034 2,2437675
214228_x_at TNFRSF4 8,910944 6,6099143 3,4565573 5,0546947
215346_at CD40 5,0572295 5,593753 6,163759 5,6992526
219211_at USP18 8,285303 7,0497055 5,0085244 3,340998
223887_at GPR132 6,7607636 4,543277 5,509192 7,0652533
226702_at LOC129607 7,0691476 5,9127674 5,569863 5,045494
227816_at LOC400572 5,9134483 6,345112 6,439589 5,0362144
231578_at GBP1 7,4657335 6,917391 3,8039267 6,1784716
232213_at PELI1 7,4485292 5,587166 5,212285 6,0972896
200628_s_at WARS 9,368469 5,516358 4,64247 5,419954
202800_at SLC1A3 6,087409 6,9154663 7,895211 3,3894732
204014_at DUSP4 8,585252 6,8769712 4,5652914 4,590569
204070_at RARRES3 5,2620735 6,591536 6,2708507 5,70036
204747_at IFIT3 8,602514 7,492393 5,6343427 3,3676317
212458_at SPRED2 5,5534644 7,490529 6,08654 4,736595
212641_at HIVEP2 6,433779 5,7410865 6,5065494 6,1581783
231779_at IRAK2 8,596796 4,4581327 5,576258 6,910897
1559777_at 5,605326 8,543147 6,0953383 4,9331293
200986_at SERPING1 9,492846 8,300841 4,229471 3,6452117
203708_at PDE4B 11,223041 4,007264 4,071895 4,2633905
204286_s_at PMAIP1 9,400938 5,3456664 4,7092376 6,7266498
205153_s_at CD40 6,071054 6,5306187 6,4112535 6,4513636
222934_s at CLEC4E 8,080958 8,404435 6,981838 2,577743
224225_s at ETV7 7,935058 6,064494 6,699362 5,50029
226474_at NOD27 5,9191046 7,2645817 5,1934004 7,0305643
227262_at HAPLN3 7,1910353 6,615262 5,1981425 7,5915074
230127_at 7,3191285 7,9864817 5,3266 6,0064306
244780_at SGPP2 8,127058 5,944443 6,879924 5,023631
1569095_at 7,318123 8,3700075 6,2253203 4,625397
201601_x_at IFITM1 7,6313257 7,573814 6,0775313 4,9257803
202643_s_at TNFAIP3 6,250073 5,1900063 6,5136204 7,95266
202760_s_at PALM2-AKAP2 9,142822 7,253937 5,7028017 5,2498336
204285_s_at PMAIP1 10,275632 5,124492 4,698287 7,937848
204363_at F3 5,9613805 7,1758485 7,078458 5,787101
208747_s_at C1S 6,224019 6,8833885 6,2983246 6,745276
209723_at SERPINB9 10,339775 5,989251 4,825143 6,5091825
214329_x_at TNFSF10 6,1703367 6,8099036 6,2552257 7,0860806
216598_s_at CCL2 7,959111 5,9420986 7,541988 5,4770517
218656_s_at LHFP 5,799256 5,6268 7,0819764 6,887301
221680_s_at ETV7 7,4525156 6,8642745 6,899107 6,077394
227677_at JAK3 5,4577355 5,5538926 6,847657 6,6245513
229450_at IFIT3 7,4833083 7,133669 7,3032265 5,366725
235574_at GBP4 8,758058 7,3968883 5,5135446 6,7058306
1554519_at CD80 7,0503464 5,5513606 6,9412417 7,924613
1555689_at CD80 8,324632 7,511187 6,2935348 6,2964225
204224_s_at GCH1 7,3962016 6,945448 5,8722777 7,290103
205114_s_at CCL3 8,18418 6,780873 7,019624 5,952113
206508_at TNFSF7 6,858301 7,575267 8,330129 4,665465
209722_s_at SERPINB9 8,607762 7,3330297 5,989785 6,4565625
213524_s_at G0S2 7,547209 7,6664352 6,440924 6,2842937
222292_at CD40 8,530226 8,246122 6,060198 6,302727
223798_at SLC41A2 9,232196 8,087529 3,90797 7,397384
242907_at GBP2 7,746566 8,2072735 5,8188763 6,584134
1555465_at MCOLN2 9,518959 8,71965 6,8508315 4,539327
200953_s_at CCND2 7,411466 6,296918 7,8509226 6,8842726
201860_s_at PLAT 8,657469 7,604994 6,370206 6,5896416
208303_s_at CRLF2 8,685696 8,653402 8,276672 3,62456
238727_at LOC440934 7,688413 7,8426127 7,593639 5,8430877
239186_at MGC39372 7,809087 6,8687167 7,7233596 6,9605627
222221_x_at EHD1 7,799284 6,630239 7,478602 7,7974253
222326_at PDE4B 8,487402 7,4390974 5,838442 8,485796
223767_at GPR84 9,556347 9,886792 8,25461 2,6693628
214022_s_at IFITM1 8,305978 9,906662 6,8351192 5,3545113
221241_s_at BCL2L14 7,763052 8,927696 7,4124217 6,0943
209037_s_at EHD1 6,202972 6,62678 6,968862 8,198914
222802_at 9,193985 9,879644 6,7598433 5,578077
234306_s_at SLAMF7 11,054989 9,386422 7,5571413 4,0329967
219584_at PLA1A 7,8830075 8,5753975 7,899431 6,5355268
232593_at LINCR 7,426831 7,1173863 7,508543 8,483282
235175_at GBP4 8,098684 8,014557 6,7604356 8,305143
238567_at SGPP2 9,60297 7,9624267 8,190959 5,9755754
202270_at GBP1 7,857806 7,5784187 7,864753 7,7254214
206058_at SLC6A12 7,0730734 7,6346216 7,868434 7,8353267
209270_at LAMB3 6,3819847 7,637574 8,068874 8,396816
223217_s_at NFKBIZ 6,579339 5,8985357 7,933263 9,0830765
230110_at MCOLN2 7,0183334 8,743037 8,322358 6,695663
235116_at TRAF1 7,515788 6,6126266 7,946375 8,608736
239196_at ANKRD22 6,893366 7,2058015 7,997893 8,679558
1557359_at LOC285758 5,7492123 6,7752757 6,428899 8,264215
202833_s_at SERPINA1 6,047883 6,2670393 8,372422 8,15069
220655_at TNIP3 4,227455 9,260285 8,14861 3,8333952
210354_at IFNG 7,0490704 9,9158745 9,995628 4,7299943
239331_at 4,9562793 7,907742 6,738407 7,5105863
表4:IL-10表达簇。依照CarmaWeb算法,所列基因具有的表达谱类似于IL-10(基因名称IL10)的表达谱,提示与IL-10功能类似的功能(数目是相对于未成熟DC的log,其中底数为2)。
12小时LPS 24小时LPS 48小时LPS
6小时LPS CD40L IFN-γ CD40L IFN-γ CD40L IFN-γ
IFN-γ对6小时 对12小时iDC 对24小时iDC 对48小时iDC
独特的ID 名称 iDC M M M M
1556300_s_a
t 0 4,323714 1,5688843 0,257391
1556378_a_a -0,12478956
t LOC401530 0 3,3170495 6 1,3912028
1556883_a_a
t LOC401528 0 4,490707 -0,14703345 2,7003522
202291_s_at MGP 0 5,571205 2,098167 0,038769286
202878_s_at C1QR1 0 3,306639 0,3353752 1,2330873
204475_at MMP1 0 5,03655 0,2590128 0,17097393
204614_at SERPINB2 0 3,4892087 2,2649622 0,08403955
205676_at CYP27B1 0 3,6301327 0,1309929 0,7396309
223287_s_at FOXP1 0 2,983712 1,7371364 0,5753527
224773_at NAV1 0 3,3603613 0,35192382 0,9197203
227812_at TNFRSF19 0 3,3981404 1,6189107 0,044692483
235042_at CMYA1 0 3,450752 0,328667 1,5720363
-0,04244264
235444_at FOXP1 0 3,0461197 6 1,3964777
241860_at STK17B 0 2,671234 0,61670065 1,4493694
1556582_at LOC440536 0 2,6907892 1,2484756 1,1807224
1564028_s_a
t FLJ40722 0 2,750025 0,67192906 1,4337206
1566480_x_a
t FLJ31795 0 2,810023 1,4379538 0,6065171
1570388_a_a
t LOC401530 0 3,911918 0,027789168 1,8137968
202877_s_at C1QR1 0 3,3630056 0,6407456 1,4440103
204602_at DKK1 0 3,921798 3,6861098 0,3208148
204932_at TNFRSF11B 0 4,2726865 3,2933848 0,21878207
215268_at MACF1 0 2,6788146 1,2724367 0,8143623
216497_at LOC120364 0 2,3947499 1,1637448 0,9242658
216867_s_at PDGFA 0 2,1128201 1,2416315 1,132686
220655_at TNIP3 0 3,474797 0,8904967 1,8436728
221870_at EHD2 0 2,571253 0,94932306 1,2732899
224771_at NAV1 0 3,8266947 1,5089604 1,4963266
238712_at 0 4,2949014 1,8307322 2,142599
239311_at DHX57 0 2,27365 1,3914022 1,0178465
1556318_s_a
t CAND1 0 2,3933206 0,9396068 1,3715496
227732_at ATXN7L1 0 3,4273734 0,6846624 2,2728565
239060_at EHD1 0 1,997289 0,32936123 1,8916218
206176_at BMP6 0 2,2903044 -0,18439728 2,1745071
207386_at CYP7B1 0 3,9868062 0,7309004 2,746021
210229_s_at CSF2 0 3,1023788 -0,1405712 2,5077276
0,00688015
215750_at KIAA1659 0 3,0428922 7 2,480364
225025_at IGSF8 0 1,905534 0,49440768 1,845558
227345_at TNFRSF10D 0 2,1187048 0,36650723 2,0576801
236738_at LOC401097 0 2,4518428 1,685922 1,7134967
242517_at GPR54 0 1,9395804 0,21328141 2,186202
228910_at CD82 0 3,5779603 1,2853656 2,5263379
229307_at ANKRD28 0 3,1102884 0,8216141 2,5533133
231832_at GALNT4 0 2,2378843 0,72167736 2,091024
37005_at NBL1 0 2,158463 1,2050192 2,0193584
227410_at FAM43A 0 3,1527455 1,5260311 2,2625198
228625_at CITED4 0 2,2035446 0,8985138 2,1428196
240432_x_at 0 2,0101619 1,3507649 2,0001297
203074_at ANXA8 0 2,754555 0,79692614 2,6273892
206009_at ITGA9 0 1,9594706 0,54135984 2,3522627
235438_at 0 4,0041165 0,70460325 3,542835
1560869_a_a
t 0 2,5460286 1,5980372 2,5416172
223525_at DLL4 0 3,5447857 0,5724994 3,72409
232090_at DNM3 0 4,302288 3,442975 2,8309567
203904_x_at CD82 0 2,0282779 1,3777583 2,4270318
225645_at EHF 0 4,428559 2,14202 3,7522347
235737_at TSLP 0 4,959613 1,5306113 4,4908447
202237_at NNMT 0 3,6071026 3,2686546 2,8587787
207433_at IL10 0 4,00954 1,7226876 3,7454016
214414_x_at HBA1 0 2,1132698 1,575519 2,6071193
224940_s_at PAPPA 0 2,786031 2,7599206 0,7074637
212730_at DMN 0 3,6943512 0,91267496 4,2896175
219874_at SLC12A8 0 2,8926702 3,182794 1,9976637
224646_x_at H19 0 2,466048 2,7163086 2,6425989
209324_s_at RGS16 0 3,3799229 2,467596 3,7553544
243788_at PHF11 0 2,1383092 2,2994013 2,1082926
202238_s_at NNMT 0 4,160124 4,563192 3,5705357
224997_x_at H19 0 2,8080769 3,1897178 3,0020173
236176_at 0 2,051894 0,7956073 3,2491539
44790_s_at C13orf18 0 2,1295624 2,4644802 2,7274456
206825_at OXTR 0 2,7509916 0,5947344 4,1146626
207442_at CSF3 0 2,2279809 1,6218964 3,2061028
216575_at 0 2,1809235 1,0562297 3,4115193
237559_at GPR55 0 2,0120387 1,6486369 3,2241027
-0,06174222
200951_s_at CCND2 0 4 2,055891 5,531777
204163_at EMILIN1 0 -0,08650574 2,116541 3,2442234
215646_s_at CSPG2 0 1,0869541 0,9552003 4,6304297
220442_at GALNT4 0 1,6785349 2,2396698 2,8896742
223194_s_at C6orf85 0 1,4545181 1,8726805 3,2625294
-0,01206030
227703_s_at SYTL4 0 6 2,8051267 3,7273147
-0,00852494
1552393_at FLJ25421 0 4 0,79469424 4,0263605
1552394_a_a
t FLJ25421 0 0,4518348 1,1220381 3,568555
1553785_at RASGEF1B 0 0,8244259 3,0841393 2,744691
1554079_at GALNTL4 0 0,80384594 2,1117246 3,2543478
1559777_at 0 1,373273 1,964059 4,745006
1562433_at FLJ10489 0 0,26058874 3,4475367 4,6152167
1568949_at PITPNC1 0 0,004084121 2,9905202 3,3454092
1569095_at 0 1,0567621 1,0456768 3,5029418
200783_s_at STMN1 0 0,2741603 1,4904935 3,2696452
202403_s_at COL1A2 0 0,83871436 2,151641 3,4803479
202431_s_at MYC 0 1,0226223 2,5720317 3,490832
202998_s_at LOXL2 0 0,4333325 1,7000065 3,451721
203108_at GPRC5A 0 1,6255034 1,8504707 4,352528
203131_at PDGFRA 0 -0,45227933 1,4600621 3,411754
203592_s_at FSTL3 0 0,14812845 2,2247574 3,3438997
-0,00912857
203980_at FABP4 0 2 1,3565757 4,341141
-0,01519909
204301_at KIAA0711 0 9 1,469634 3,7521276
204411_at KIF21B 0 0,99417245 2,3202088 3,2713737
204619_s_at CSPG2 0 1,7562126 1,4990444 4,1206384
204620_s_at CSPG2 0 1,6183093 1,011578 4,371935
204879_at PDPN 0 -0,15425473 0,95934194 3,9027925
204904_at GJA4 0 0,30981743 2,2777586 3,7517166
205100_at GFPT2 0 1,5140616 1,8844032 5,630774
205289_at BMP2 0 0,60859656 1,5663037 3,305952
205290_s_at BMP2 0 0,07147631 1,7073456 4,3988748
-0,00498334
205826_at MYOM2 0 2 3,2177415 6,163263
205861_at SPIB 0 0,7059429 2,4294124 2,704671
-0,01041656
205898_at CX3CR1 0 92,8846204 5,349967
206027_at S100A3 0 0,13657278 1,5692319 3,4727564
206090_s_at DISC1 0 -0,8741116 1,8682606 5,05058
206729_at TNFRSF8 0 0,27889317 2,6180744 5,8870077
206741_at LOC51066 0 0,47887027 2,5547576 5,7319098
-0,00132160
206859_s_at PAEP 0 7 1,3401538 3,7394311
207510_at BDKRB1 0 2,0661907 3,4934416 3,781754
209325_s_at RGS16 0 1,5113075 2,5088499 3,8291175
210095_s_at IGFBP3 0 0,21584912 2,5991304 3,2872534
211372_s_at IL1R2 0 -0,6165594 1,6310264 3,7544057
211571_s_at CSPG2 0 0,91350543 0,87646186 5,6404405
211596_s_at LRIG1 0 3,8467457 2,826491 5,69805
211597_s_at HOP 0 0,1277425 1,6993432 6,786917
212143_s_at IGFBP3 0 -0,19099335 3,7475343 4,0994177
212444_at 0 1,1151347 1,3201189 3,6738355
213139_at SNAI2 0 0,001255514 3,9536047 5,2359695
215495_s_at SAMD4 0 -0,6807276 2,1140902 4,5931683
218574_s_at LMCD1 0 0,004399216 0,005991654 4,832267
218975_at COL5A3 0 1,8476033 3,2505863 4,3477015
219168_s_at PRR5 0 0,3802334 1,883373 5,4903607
219181_at LIPG 0 0,034028087 1,7650458 4,60227
221731_x_at CSPG2 0 1,1740024 1,1199198 4,3726726
224950_at PTGFRN 0 0,2711382 2,548231 4,0687737
225571_at LIFR 0 1,2333707 2,1292431 2,7829444
226621_at FGG 0 0,91611135 2,2092934 3,1336222
227256_at USP31 0 0,66912067 1,812497 3,3547237
228245_s_at OVOS2 0 -0,6036949 1,9032807 5,509818
228367_at ALPK2 0 1,9285325 1,4045739 3,7516391
228854_at 0 0,013371324 1,4183841 4,6363435
229247_at FLJ37440 0 9,65E-04 0,14736369 5,156355
229622_at FLJ43374 0 0,15105823 2,228979 3,3192601
230233_at RASGEF1B 0 0,44206667 2,0026255 4,1667137
231496_at FCAMR 0 0,0146722 3,7155752 2,9080575
-0,00254589
231867_at ODZ2 0 3 -9,53E-04 4,766165
232739_at SPIB 0 -0,03394005 2,8278906 2,699333
235100_at 0 1,1068798 2,099443 3,164012
237344_at 0 0,14466353 0,72832346 5,1735163
-0,03585563
239808_at PITPNC1 0 6 0,69854295 4,18631
240770_at PRP2 0 0,35803238 1,2584826 3,8403585
242691_at 0 0,042725943 2,1449049 3,0019956
-0,00170695
40687_at GJA4 0 6 2,8572736 4,300132
41469_at PI3 0 0,08784416 3,8997574 2,944381
47069at PRR5 0 -0,30954257 1,9246503 5,094549
52255_s_at COL5A3 0 2,9138856 3,5991492 5,075999
223503_at DKFZP566N034 0 0,001206623 0,010989565 4,3298063
在本DNA微阵列中,还鉴定出在LPS/IFN-γ或CD40L/IFN-γ信号传导后在DC中诱导,且牵涉调节基因IL-10、TSLP、INDO、IL2RA、CSF-2和CSF-3(已知它们都具有免疫抑制效果)的基因。为了鉴定免疫调节的潜在的主开关,用Pathway Studio软件使用Resnet 5(第1.2版,2007年1月)(自PubMed和44份开架阅览期刊获得的哺乳动物途径和分子相互作用的数据库)产生那些基因的调节物的网络。通过将来自差别活化DC的微阵列数据上载至调节网络,然后可以选择使DC成熟中诱导的潜在主调节物(表5)。
表5:免疫调节的主开关。
免疫调节 Affymetrix索引
STAT6 IL10 201332_s_at
LITAF IL10 200706_s_at
IL10,INDO,IL2RA,
STAT1 CSF3 232375_at
IRF4 IL10,CSF2 204562_at
IRF1 IL10,INDO,IL2RA 202531_at
IRF2 未知 203275_at
REL IL10 206035_at
NFKB1 IL10,IL2RA 239876_at
STAT3 IL2RA 235680_at
RELA IL10,IL2RA 209878_s_at
JUNB IL10,CSF2 201473_at
CEBPB IL10,CSF3 212501_at
TBX21 IL10,IL2RA 220684_at
JUN CSF2 201465_s_at
STAT5B IL2RA 205026_at
STAT5A IL2RA,CSF2 203010_at
STAT4 未知 206118_at
ETV6 CSF2 205585_at
EGR1 IL2RA 227404_s_at
NFATC1 IL10 210162_s_at
CREB1 CSF2 214513_s_at
JAK1 TSLJP,CSF3 1552611_a_at
JAK3 IL10,TSLP,CSF2 227677_at
LYN IL10 202626_s_at
MAPKA
PK2 IL10201460_at
MAP21 IL10,IL2RA,CSF2 202670_at
CD274 未知 223834_at
PDL2 IL10 224399_at
PTGR4 IL10,IL2RA 204896_s_at
ITGAX IL10 210184_at
ADORA
2A IL10 205013_s_at
SerpinA1 IL10 202833_s_at
SerpinE1 CSF2 202627_s_at
AREG CSF2 205239_at
OSM CSF3,CSF2 230170_at
SOCS3 未知 206359_at
IFNG INDO,IL2RA 210354_at
图9显示了使用RNA干扰敲除DC中在表达谱分析中鉴定的靶分子的表达后关于改善的增殖响应的例子。显示了,展现出与IL-10、IDO类似的DC表达动力学,或者属于免疫调节的主开关簇的基因牵涉负调节免疫抑制反馈环。设计与那些使用RNA干扰敲除IL-10或IDO表达的实验类似的实验。用对来自表3、4、或5的基因特异性的siRNA转染DC。图9中显示了基因MAPKAPK2、IRF2、PHF11、IRF4、JAK1、CEBPB、和ETV6。通过用LPS/IFN-g暴露6小时来启动成熟后,将经过工程化改造的DC与异基因T淋巴细胞共培养6天。用CFSE(即一种进入细胞、结合蛋白质,并被保留在细胞内部的荧光染料)标记异基因T淋巴细胞;洗掉过量的CFSE。随着每次细胞分裂,T淋巴细胞的荧光强度被平分,这容许在共培养第6天时评估T淋巴细胞增殖。作为对照,使用未转染的DC或用对照siRNA转染的DC。经工程化改造的DC刺激异基因T淋巴细胞的能力改善提供关于siRNA靶向的基因牵涉负调节反馈环的证据。此外,这指明与常规的免疫治疗剂相比,为了敲除此类基因而遗传工程化改造的DC免疫药物会具有改善的治疗效果。
结论
本发明提供了如下证据,即DC免疫药物的特征可以通过遗传工程进行调控。证明了免疫刺激分子在DC中的过表达以及阻断免疫抑制性分子导致增强的免疫刺激能力。这可以获得作为DC癌症疫苗或抗感染DC免疫药物的应用,其中给DC加载肿瘤衍生的抗原或自微生物衍生的抗原,将其暴露于成熟刺激物,并进行工程化改造,如所描述的。另外,本文所呈现的数据暗示可以如下设计免疫抑制性DC药物,即敲除免疫刺激分子和过表达免疫抑制性分子。此类抑制性DC免疫药物可以在变态反应或自身免疫中,而且在移植药物中具有应用,以便使移植接收者的免疫系统对所移植的组织变成无反应性(tolerise)。
Claims (20)
1.包含部分成熟的树突细胞的药物制剂,其通过包括下列步骤的方法可获得:
a)提供未成熟的树突细胞或其前体细胞或部分成熟的树突细胞,所述部分成熟的树突细胞如下可获得,即使未成熟的树突细胞与至少一种树突细胞成熟剂接触来生成部分成熟的树突细胞(半成熟的DC,smDC),如以其分泌IL-12的能力所定义的,
b)操作步骤a)的所述细胞,特别是步骤a)的释放IL-12的所述部分成熟的树突细胞(半成熟的DC,smDC),以便
(i)过表达能够使以连续的IL-12分泌表征的树突细胞的T淋巴细胞刺激能力维持至少24小时,优选至少48小时,且选自CD40L的至少一种免疫分子,其通过导入编码所述至少一种分子的核酸分子来实现;和/或
(ii)抑制或阻止在暴露于初级成熟剂诸如LPS/IFN-γ的树突细胞内起作用或自暴露于初级成熟剂诸如LPS/IFN-γ的树突细胞释放,并且选自白介素10(IL-10)和吲哚胺2,3-加双氧酶(IDO)的至少一种T-淋巴细胞抑制分子,诸如表3、4和/或5中给出的至少一种基因的表达,其如下实现,即敲除编码所述至少一种T-淋巴细胞抑制分子的基因或其片段或导入用以抑制或阻止在所述树突细胞内有活性或从所述树突细胞投递至T细胞的至少一种T-淋巴细胞抑制分子的表达的核酸分子,优选核糖核酸分子,并
c)任选地,添加物质来将树突细胞的前体细胞转化成树突细胞。
2.依照权利要求1的制剂,其特征在于所述未成熟树突细胞的前体,特别是单核细胞或造血干细胞,或所述未成熟的树突细胞获自皮肤、脾、骨髓、胸腺、淋巴结、脐带血、或外周血。
3.依照权利要求1或2的制剂,其特征在于所述至少一种树突细胞成熟剂选自下组:TLR激动剂诸如热灭活的或经福尔马林处理的卡介苗(BCG),优选BCG的细胞壁成分、BCG衍生的脂阿拉伯甘露聚糖或BCG成分,自大肠杆菌衍生的脂多糖(LPS)、或灭活的革兰氏阳性或革兰氏阴性微生物、咪唑喹啉化合物,优选咪唑喹啉-4-胺化合物,特别是4-氨基-2-乙氧基甲基-x-二甲基-1H-咪唑[4,5-c]喹啉-1-乙醇或1-(2-甲基丙基)-1H-咪唑[4,5-c]喹啉-4-胺,或其衍生物,合成的双链多核糖核苷酸,优选多聚I:C、天然的双链RNA或RNA病毒或RNA片段、或合成的类似物、或包含未甲基化的CpG基序的合成的或天然的核酸分子、细胞因子组合,优选肿瘤坏死因子α(TNF-α)、IL-1、IL-6或前列腺素E6、CD40L,优选重组CD40L或包含CD40L域的融合蛋白,或遗传工程化改造成表达CD40L的原代细胞或细胞系,或在生理学上上调CD40L表达的活化T淋巴细胞诸如T淋巴细胞。
4.依照权利要求1至3任一项的制剂,其特征在于在所述操作步骤b)前,使所述未成熟的树突细胞与所述至少一种树突细胞成熟剂接触至少2小时,优选至少4小时,更优选至少6小时,特别是至少12小时,最多24小时,以便给出部分成熟的树突细胞。
5.依照权利要求1至4任一项的制剂,其特征在于所述未成熟的或所述部分成熟的树突细胞加载有至少一种抗原。
6.依照权利要求5的制剂,其特征在于所述至少一种抗原选自下组:
a)肿瘤抗原、病毒抗原、细菌抗原、或任何其它人微生物或寄生物病原体;或
b)引起变态反应的环境抗原、启动的免疫应答所针对的且引起疾病的自身抗原、或移植抗原。
7.用于生成树突细胞的方法,包括下列步骤:
a)提供未成熟的树突细胞,
b)使所述未成熟的树突细胞与至少一种树突细胞成熟剂接触来生成部分成熟的树突细胞(半成熟的DC,smDC),如以其分泌IL-12的能力所定义的,并
c)操作步骤b)的释放IL-12的所述部分成熟的树突细胞(半成熟的DC,smDC),以便
(i)过表达能够使以连续的IL-12分泌表征的所述树突细胞的T淋巴细胞刺激能力维持至少24小时,优选至少48小时,且选自CD40L的至少一种免疫分子,其通过导入编码所述至少一种分子的核酸分子来实现;和/或
(ii)抑制或阻止在暴露于初级成熟剂诸如LPS/IFN-γ的所述树突细胞内起作用或自暴露于初级成熟剂诸如LPS/IFN-γ的树突细胞释放,并且选自白介素10(IL-10)和吲哚胺2,3-加双氧酶(IDO)的至少一种T-淋巴细胞抑制分子,诸如表3、4和/或5中给出的至少一种基因的表达,其如下实现,即敲除表达所述至少一种T-淋巴细胞抑制分子的基因或其片段或导入用以抑制或阻止在所述树突细胞内有活性或从所述树突细胞投递至T细胞的至少一种T-淋巴细胞抑制分子的表达的核酸分子,优选核糖核酸分子。
8.依照权利要求7的方法,其特征在于所述未成熟树突细胞的前体或所述未成熟的树突细胞或部分成熟的树突细胞获自皮肤、脾、骨髓、胸腺、淋巴结、脐带血、或外周血。
9.依照权利要求7或8的方法,其特征在于所述至少一种树突细胞成熟剂选自下组:TLR激动剂诸如热灭活的或经福尔马林处理的卡介苗(BCG),优选BCG的细胞壁成分、BCG衍生的脂阿拉伯甘露聚糖或BCG成分,自大肠杆菌衍生的脂多糖(LPS)、或灭活的革兰氏阳性或革兰氏阴性微生物、咪唑喹啉化合物,优选咪唑喹啉-4-胺化合物,特别是4-氨基-2-乙氧基甲基-x-二甲基-1H-咪唑[4,5-c]喹啉-1-乙醇或1-(2-甲基丙基)-1H-咪唑[4,5-c]喹啉-4-胺,或其衍生物,合成的双链多核糖核苷酸,优选多聚I:C、天然的双链RNA或RNA病毒或RNA片段、或合成的类似物、或包含未甲基化的CpG基序的合成的或天然的核酸分子、细胞因子组合,优选肿瘤坏死因子α(TNFα)、IL-1、IL-6或前列腺素E6、CD40L,优选重组CD40L或包含CD40L域的融合蛋白,或遗传工程化改造成表达CD40L的原代细胞或细胞系,或在生理学上上调CD40L表达的活化T淋巴细胞诸如T淋巴细胞。
10.依照权利要求7至9任一项的方法,其特征在于在所述操作步骤c)前,使所述未成熟的树突细胞与所述至少一种树突细胞成熟剂接触至少2小时,优选至少4小时,更优选至少6小时,特别是至少12小时,最多24小时。
11.依照权利要求7至10任一项的方法,其特征在于所述未成熟的或所述部分成熟的树突细胞加载有至少一种抗原。
12.依照权利要求11的方法,其特征在于所述至少一种抗原选自下组:
a)肿瘤抗原、病毒抗原、细菌抗原、或任何其它人微生物或寄生物病原体;或
b)引起变态反应的环境抗原、启动的免疫应答所针对的且引起疾病的自身抗原、或移植抗原。
13.通过依照权利要求7至12任一项的方法可获得的树突细胞。
14.包含依照权利要求13的树突细胞的药物组合物。
15.依照权利要求13的树突细胞或依照权利要求1至6任一项的制剂用于制备药物的用途,所述药物用于治疗和/或预防癌症和/或微生物或寄生物感染;或用于治疗和/或预防变态反应、自身免疫性疾病、或干细胞或器官移植排斥。
16.依照权利要求15的用途,其特征在于在放射疗法和/或抗肿瘤或抗微生物化学疗法、或目的在于治疗变态反应、自身免疫性疾病、或干细胞或器官移植排斥的任何疗法前、同时、或之后对个体施用所述药物。
17.依照权利要求13的树突细胞或依照权利要求1至6任一项的制剂用于制备药物的用途,所述药物用于治疗和/或预防由免疫系统针对环境抗原诸如变应原,或针对自身免疫性疾病过程中的自身抗原的病理学反应过度引起的免疫学疾病。
18.依照权利要求17的用途,其特征在于在目的在于治疗或预防变态反应或自身免疫性疾病的其它药征前、同时、或之后对个体施用所述药物。
19.依照权利要求13的树突细胞或依照权利要求1至6任一项的制剂用于制备药物的用途,所述药物用于治疗和/或预防异基因干细胞移植(优选在治疗血液学恶性肿瘤中使用)的免疫学排斥,或者用于治疗和/或预防异基因器官移植的排斥。
20.依照权利要求19的用途,其特征在于在目的在于治疗或预防所述异基因干细胞或器官移植排斥的其它药征前、同时、或之后对个体施用所述药物。
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US20080145931A1 (en) * | 2004-10-07 | 2008-06-19 | Argos Therapeutics, Inc. | Mature Dendritic Cell Compositions and Methods of Culturing Same |
US8440610B2 (en) * | 2004-11-12 | 2013-05-14 | Massachusetts Institute Of Technology | Mapkap kinase-2 as a specific target for blocking proliferation of P53-defective cells |
EP2072617A1 (en) * | 2007-12-12 | 2009-06-24 | Trimed Biotech GmbH | Method for producing dendritic cells |
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CN106687583A (zh) * | 2014-06-23 | 2017-05-17 | Jw可瑞基因株式会社 | 用于制备具有增加的特异性基因表达的树突细胞的方法和包含使用所述方法制备的树突细胞的用于治疗或预防自身免疫性疾病的组合物 |
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CN108291204A (zh) * | 2015-09-15 | 2018-07-17 | 西北生物治疗药物公司 | 涉及用于患有晚期癌症的受试者的经活化树突状细胞组合物和免疫治疗处理的方法 |
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CN105219720A (zh) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | 一种用于激活肝癌特异性免疫反应的试剂盒 |
CN114032212A (zh) * | 2022-01-10 | 2022-02-11 | 北京荟科柘生物科技有限公司 | 一种提高抗原呈递t细胞并提高t细胞杀伤效率的dc细胞制备方法及其应用 |
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CA2709209C (en) | 2019-05-21 |
EA201000972A1 (ru) | 2010-12-30 |
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EA023106B1 (ru) | 2016-04-29 |
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CA2709209A1 (en) | 2009-06-18 |
AU2008334914A1 (en) | 2009-06-18 |
JP2011506372A (ja) | 2011-03-03 |
NZ585898A (en) | 2012-01-12 |
US9303246B2 (en) | 2016-04-05 |
ZA201003193B (en) | 2011-10-26 |
AU2008334914B9 (en) | 2013-09-26 |
CN101896601B (zh) | 2014-09-03 |
US20100285072A1 (en) | 2010-11-11 |
WO2009074341A1 (en) | 2009-06-18 |
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