CN1172718C - 树突状细胞刺激因子 - Google Patents
树突状细胞刺激因子 Download PDFInfo
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- CN1172718C CN1172718C CNB96197351XA CN96197351A CN1172718C CN 1172718 C CN1172718 C CN 1172718C CN B96197351X A CNB96197351X A CN B96197351XA CN 96197351 A CN96197351 A CN 96197351A CN 1172718 C CN1172718 C CN 1172718C
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Abstract
Flt3-配体可以用于由造血祖细胞和干细胞产生大量树突状细胞。Flt3-配体可以用于在体内放大免疫应答,在体外扩增树突状细胞。然后可将这类树突状细胞用于向幼稚T细胞提呈肿瘤、病毒或其它抗原,因而可以用作疫苗的佐剂。
Description
发明领域
本发明涉及一种树突状细胞刺激因子,涉及增强体内免疫应答的方法,涉及体外扩增树突状细胞的方法,涉及纯化树突状细胞的制剂,还涉及可用于调控由T细胞或B细胞介导的免疫应答的树突状细胞群。
发明背景
疫苗接种的目的是通过产生足够的抗体水平和准备好在再次接触抗原时能够快速扩增的细胞群来提供有效的免疫性。疫苗接种时与抗原的首次接触必须不能对受者造成伤害,所以通常使用致病性缺陷型抗原。
主动免疫方法中的常见难题是,疫苗抗原未呈现足够的免疫原性以引发强烈的免疫应答,所以不能够在再次受到相同抗原攻击时提供足够的保护水平。此外,某些抗原只能激发弱的细胞介导的或抗体应答。对于许多抗原,需要同时激发强体液应答和强细胞介导应答。
长期以来,研究者已经试验了许多不同的化合物来提高疫苗的免疫原性。疫苗的免疫促进剂,又称佐剂,是由促进对疫苗产生强免疫应答的物质组成的组合物。此外,某些新的重组抗原免疫原性较弱,因而需要更强的佐剂。疫苗佐剂具有不同的作用方式,同时影响免疫应答的量和质。这些作用方式可以是激活T细胞、起存储作用和改变淋巴细胞循环来使这些细胞保留在排液性淋巴结(draininglymph nodes)中。它们还可以将抗原集中在免疫部位,由此使得抗原特异性T细胞和B细胞更有效地与抗原提呈细胞反应。它们还能够刺激T细胞的增殖和分化,并作用于B细胞,例如增强不同的Ig型(isotype)的产生。而且,佐剂还能够刺激和影响抗原提呈细胞尤其是巨噬细胞的行为,使它们更有效地向T细胞和B细胞提呈抗原。
树突状细胞是一种较少的异源细胞群,具有不同的形态和广泛的组织分布。在此,提供参考的为Steinman,R.M,《免疫学评论年刊》(Annu.Rev.Immunol.)9:217-296(1991)论述了树突状细胞系统及其在免疫原性中的作用。树突状细胞具有特殊的细胞表面,其特征在于具有细胞表面标记CD1a+,CD4+,CD14-CD86+,CD11c+,DEC-205+。CD14+或HLA-DR+。树突状细胞具有很强的致敏MHC限制性T细胞的能力,并为向T细胞原位提呈抗原,不论是在T细胞发育期间的自身抗原还是免疫期间的外源抗原,提供了有效的途径。所以,人们对在体外将树突状细胞用作肿瘤或传染病疫苗的佐剂越来越感兴趣。参见例如Romani等,《实验医学杂志》(J.Exp.Med.)180:83(1994)。由于树突状细胞在外周血中出现频率较低,与淋巴器官的接触有限,以及树突状细胞的最终分化形态,树突状细胞作为免疫促进剂的用途受到了限制。树突状细胞来自CD34+骨髓祖细胞,细胞因子粒细胞-巨噬细胞集落因子(GM-CSF)(sargramostim,Leukine,Immunex Corporation,Seattle,Washington),肿瘤坏死因子-α(TNF-α),c-kit配体(又称干细胞因子(SCF),青灰因子(Steel Factor,SF)或肥大细胞生长因子(MGF))和白细胞介素-4能够促进树突状细胞的增殖和成熟。
因此一种能在体内或体外刺激功能成熟的树突状细胞大量增殖的制剂必有更大的重要性。
发明概述
已知flt-3配体(“flt-3配体”或“flt-L”)能影响造血干细胞和祖细胞。出人意料的是,flt3-配体还被发现能够强烈刺激由下游细胞或中间细胞,例如髓样前体细胞、单核细胞、巨噬细胞、B细胞和来自CD34+骨髓祖细胞和干细胞的树突状细胞。本发明是关于一种体内激发树突状细胞,体外扩增树突状细胞的方法和纯化的树突状细胞制剂。本发明的纯化树突状细胞制剂有可能用作疫苗的佐剂。本发明的实施方式中还包括利用被flt3-配体活化的树突状细胞来制备抗原特异性T细胞的方法。
本发明使用有效量的flt3-配体在体内,例如在患者的外周血、组织或器官内增加或汇集中间细胞的数量。虽然本发明涉及用flt3-配体从CD34+细胞增殖大量这类下游细胞(down steam cell)和中间细胞(intermediate cell)(例如髓样细胞、单核细胞和巨噬细胞),但主要侧重于树突状细胞。通过增加病人的树突状细胞量,可以利用这些细胞自身来向T细胞提呈抗原。例如,抗原可能是原先已经存在于患者体内的抗原,例如肿瘤抗原、或者细菌或病毒抗原。所以,flt3-配体可以用来增加体内的树突状细胞数量,由此加强患者对已存在的抗原的免疫应答。或者,为了进行免疫,可以在给患者使用抗原之前、同时或之后使用flt3-配体。这样,作为疫苗佐剂,flt3-配体能够在体内增殖大量树突状细胞和其它中间细胞,从而更有效地提呈抗原。总体应答是一次对抗原更强的、更好的免疫应答以及更有效的免疫。
本发明还提供了一种在体外增殖大量树突状细胞的方法。在收集患者的CD34+造血祖细胞和干细胞之后,可以使用flt3-配体在体外扩增这些细胞(又称体外扩增),并驱使这些CD34+细胞分化成淋巴或髓样细胞系的树突状细胞。然后可以将收集的树突状细胞给患者使用,产生对抗原的更强、更好的免疫应答。或者,可将生成的树突状细胞用作疫苗佐剂,可在给以抗原之前、同时或之后给予。
本发明还提供了一种在体外增殖大量抗原提呈树突状细胞的方法。在收集患者的CD34+造血祖细胞和干细胞之后,可以使用flt3-配体在体外扩增这些细胞,并驱使这些CD34+细胞分化成淋巴或髓样细胞系的树突状细胞。然后将收集到的树突状细胞与抗原接触,并令其在体外加工和提呈抗原(该过程在本领域内有时又称“抗原脉冲”(antigenpulsing))。另一种制备提呈抗原的树突状细胞的方法是用编码抗原特异性多肽的基因转染树突状细胞。在树突状细胞表达该抗原后,该抗原提呈树突状细胞可被用于患者。
本发明还提供了体外制备抗原特异性T细胞的方法。在按上文所述体外制备大量抗原提呈性树突状细胞的方法之后,收集后的抗原提呈性树突状细胞被用于由已采自患者的幼稚T细胞产生抗原特异性T细胞。在向已增殖的T细胞提呈了足够的抗原之后,可将该抗原特异性T细胞用于患者。
本发明还提供了一种在有传染病的患者中放大免疫应答的方法,该方法包括给患者使用用量足以提高患者的树突状细胞数量的flt3-配体的步骤。
本发明还提供了一种在有癌或肿瘤的患者中放大免疫应答的方法,该方法包括一步给患者使用用量足以提高患者的树突状细胞数量的flt3-配体的步骤。该方法提供了一种放大患者肿瘤特异性免疫应答的手段。
一种增强患者自身免疫耐受性的方法,该方法包括给患者使用用量足以提高患者的树突状细胞数量的flt3-配体的步骤。本发明还包括促进移植物和被移植的组织和器官存活的方法。
本发明的方法还包括在使用flt3-配体之后或同时使用有效量的细胞因子。这些细胞因子包括但不限于白细胞介素(“ILS”)IL-3和IL-4,选自粒细胞巨噬细胞集落刺激因子(“GM-CSF”)或GM-CSF/IL-3融合体的集落刺激因子(“CSF”),或TNF-α或c-kit配体等其它细胞因子。
本发明还包括一种树突状细胞扩增培养基,其中包含细胞生长培养基、自体血清和单用flt3-配体或其与以上所列细胞因子的混合物。
详细说明
本发明涉及用flt3-配体从CD34+造血祖细胞和干细胞增殖大量中间细胞型。这些中间细胞型包括髓样细胞、单核细胞、巨噬细胞、B细胞和树突状细胞。体内并不天然存在大量的这类中间细胞型,但是可以通过使用flt3-配体来增殖。这种总体上的细胞增加能够放大宿主体内对抗原的免疫应答。本发明的另一种实施方式是分离和使用这些中间细胞型作为抗原提呈细胞,或将它们用作疫苗佐剂。虽然本发明集中论述了有关树突状细胞的实施方式,但是也适用于髓样细胞、单核细胞和巨噬细胞型。
在本文中,“flt3-配体”指在此引为参考的美国专利5,554,512,EP0627487A2和WO94/28391中所述的一类多肽。人flt3-配体cDNA已于1993年8月6日保藏在美国典型培养物保藏中心,Rockvill,Maryland,USA(ATCC),保藏号为ATCC69382。保藏是按照布达佩斯条约的条款进行的。flt3-配体可以按照以上引用的文献中所述的方法来制备。
“IL-3”指在此参考的美国专利5,108,910中所述的白细胞介素-3多肽类。这类多肽包括氨基酸序列与在此参考的例如EP专利公开275,598和282,185中所述的天然人白细胞介素-3的氨基酸序列基本相同的类似物。“IL-3”还包括至少表现出某些与天然人IL-3分子相同的生物活性的类似物和等位基因。EP专利公开282,185中例举的IL-3的类似物。其它形式的IL-3包括人IL-3[Pro8Asp15Asp70]、人IL-3[Ser8Asp15Asp70]和人IL-3[Ser8]。适用于本发明的编码人IL-3蛋白的DNA序列可按保藏号ATCC67747向美国典型培养物保藏中心(ATCC)获取。本发明所用的、在括弧中加注了氨基酸序列的名称指出了与天然人型不同的氨基酸。例如人IL-3[Ser8Asp15Asp70]指人IL3蛋白中第8位氨基酸被丝氨酸残基取代,第15位氨基酸被天冬氨酸残基取代,第70位氨基酸被天冬氨酸残基取代。
“IL-4”指在此列为参考的Mosley等在《细胞》(Cell)59:335(1989),Idzerda等在《实验医学杂志》(J.Exp.Med.)171:861(1990)和Galizzi等在《世界免疫学》(Intl.Immunol.)2:669(1990)中所述的一类多肽。这类IL-4多肽包括如下类似物,即具有与Mosley等、Idzerda等和Galizzi等所述的天然人IL-4氨基酸序列基本相同的氨基酸序列,具有生物学活性即能够结合IL-4受体,转导通过结合IL4受体引发的生物学信号或者与抗IL-4抗体交叉反应。“IL-4”还包括足以保留天然人IL-4生物活性的人IL-4分子的类似物。
本文中,“GM-CSF”指在此引为参考的美国专利5,108,910和5,229,496中所述的一类蛋白质。这类蛋白质包括如下类似物,即具有与天然人GM-CSF氨基酸序列基本相同的氨基酸序列(例如公开可得到的ATCC53157或ATCC39900),并具有生物活性,即能够结合GM-CSF受体,转导通过结合GM-CSF受体引发的生物学信号或与抗GM-CSF抗体交叉反应。氨基酸序列公开在例如Anderson等《美国科学院院报》(Proc.Natl.Acad.Sci.,USA)82:6250(1985)。市售GM-CSF可以向Immunex Corp.,Seattle,WA购买(sargramostim,Leukine)。“GM-CSF”还包括美国专利5,108,910和5,229,496所述、足以保留天然人GM-CSF生物学活性的天然人GM-CSF类似物。在此列为参考的EP专利公开212914和WO89/03881中例举了GM-CSF类似物。也可以使用其它GM-CSF类似物来构建与IL-3的融合蛋白。一段编码优选的,有已切除可能的糖基位点的GM-CSF蛋白质的DNA序列可以按保藏号ATCC67231向ATCC获取。
“GM-CSF/IL-3融合蛋白”指GM-CSF与IL-3的C末端与N末端融合。该融合蛋白是已知的,在此列为参考的美国专利5,199,942,5,108,910和5,073,627中有所说明。较好的融合蛋白是美国专利5,199,942中的PIXY321。
“c-kit配体”又称肥大细胞生长因子(MGF),青灰因子或干细胞因子(SCF),是指在此列为参考的EP423,980中所述的一类多肽,该申请要求了于1990年10月1日申请的美国专利申请580,701的优先权。这类c-kit配体多肽包括有与EP423,980中所述的天然人c-kit配体氨基酸序列基本相同氨基酸序列的类似物,并具有生物学活性即能够结合c-kit配体、转导由结合c-kit配体引发的生物学信号,或者与抗c-kit配体抗体交叉反应。“c-kit配体”还包括足以保留天然人c-kit配体的生物学活性的天然人c-kit配体分子的类似物。
“佐剂”指提高、放大或增强宿主对疫苗抗原的免疫应答的物质。
造血干细胞和祖细胞的“体外扩增”是在此列为参考的美国专利5,199,942中所述的方法。简而言之,该方法包括:(1)从患者的外周血收获物或骨髓抽出物采集CD34+造血干细胞和祖细胞;和(2)在体外扩增这些细胞。除了专利5,199,942中所述的细胞生长因子,还可以使用诸如flt3-配体、IL-1、IL-3,c-kit配体等其它因子。
“免疫原性”指诱导免疫应答的免疫原或抗原的相对效力。
“基本相同”指变异体氨基酸序列与天然氨基酸序列的一致性至少80%为佳,至少90%更好。一致性百分比可以例如通过使用Devereux等所述的威斯康星大学遗传学计算机组(University of Wisconsin Genetics ComputerGroup(UWGCG))的6.0版GAP计算机程序比较序列信息来测定。GAP程序利用了经Smith和Waterman(《高级应用数学》(Adv.Appl.Math)2:482,1981)修改后的Needleman和Wunsch(《分子生物学杂志》(J.Mo1.Biol.)48:443,1970)的对齐法。GAP程序的默认参数包括:(1)用于核苷酸的unary比较矩阵(1表示一致,0表示不一致)和Gribskov和Burgess,《核酸研究》(Nucl.Acids.Res.)14:6745,1986的重量(weighted)比较矩阵,正如Schwartz和Dayhoff编的《蛋白质序列和结构图集》(Atlas of Protein Sequence and Structure),National Biomedical Research Foudation,pp.353-358,1979;和(2)每个孔隙扣3.0,每个孔隙内的每个标记加扣0.10;(3)最后的孔隙不扣分。变异体可能包含保守性取代序列,即给定的氨基酸残基被理化性质相同的残基所取代。保守性取代例如脂肪族残基的相互取代,例如Ile、Val、Leu或Ala的相互取代;或者极性残基的相互取代,例如Lys与Arg之间;Glu和Asp之间;或Gln和Asn之间。其它这类保守性取代,例如具有相同疏水性的整个区域的取代,也是众所周知的。本发明还包括天然发生的变异体。这类变异体例如因mRNAJ剪切改变或天然蛋白的蛋白酶剪切产生的蛋白质,但是它们保留了天然的生物活性。
在本文中,“疫苗”指包含某种无害形式的抗原的生物体或物质。疫苗的目的是激发免疫保护性应答。疫苗可以是重组的或非重组的。当疫苗被接种到无免疫宿主体内时,它将激发对该生物体或物质的主动免疫,但是不会引起疾病。疫苗可以是例如类毒素形式的,即被脱毒但是保留了其主要免疫原性决定基的毒素;或者是被灭活的生物体,例如仿寒(typhoid)、霍乱和脊髓灰质炎;或者是减毒生物体,即有活性但无毒性的病原体,或者可以是由这些生物体编码的抗原,或者是活的肿瘤细胞或肿瘤细胞上的抗原。
已经有多种已知的细胞选择技术可用于从细胞群中鉴定和分离出CD34+造血干细胞和祖细胞。用于鉴定和选择这些细胞类型的方法和材料都是已知的。例如,可以用单克隆抗体结合干细胞或祖细胞上的标记蛋白或表面抗原蛋白。这些造血干细胞的标记或细胞表面抗原包括CD34和Thy-1。在一种方法中,抗体被固定在某种表面例如玻璃珠上,然后与可能含有干细胞的细胞悬浮混合物接触。这就可以令抗体结合干细胞并将之固定在玻璃珠上。或者,可以将抗体与细胞混合物一起孵育,然后令形成的结合物与对抗体-细胞复合物具有亲和性的表面接触。不需要的细胞和细胞物质被去除,由此得到较纯的干细胞群。具有CD34标记的干细胞或祖细胞仅占骨髓中单核细胞的约1%至3%。外周血中的CD34+干细胞或祖细胞约比骨髓中的少10倍至100倍。
由于本发明的特殊性,选择合适的干细胞或祖细胞选择方法将取决于欲分离细胞合适的表型。造血干细胞是根据其物理特性来选择的,这些特性例如表达与膜结合的flt3受体,或者具有下述的细胞标记:CD34或Thy-1。在此列为参考的美国专利4,714,680中描述了识别所有这类抗原的单克隆抗体(抗-My-10),抗-CD34可以购自Becton Dickinson,Franklin Lakes,NJ,抗-Thy-1单克隆抗体可以方便地利用在此列为参考的Dalchau等在《实验医学杂志》(J.Exp.Med.)149:576(1979)中所述的方法产生。也可以使用flt3受体结合蛋白,例如抗-flt3单克隆抗体或flt3-配体。令细胞结合蛋白与集得的细胞混合物接触,培养足够的时间,以便所需的细胞与细胞结合蛋白结合。
另一种选择静息干细胞的方法是利用抗代谢药例如5-氟尿嘧啶(5-FU)或烷基化剂例如4-羟基环磷酰胺(4-HC)在正在分裂、谱系更明确的细胞类型中诱导细胞死亡。通过加入对干细胞作用很小或没有作用的生长因子来刺激非静息细胞的增殖和分化,由此导致非干细胞的增殖和分化,使得它们更易于受到5-FU或4-HC的细胞毒性作用。参见在此引为参考Berardi等,《科学》(Science)267:104(1995)。
可以利用例如亲和层析、抗体包裹的磁性珠、或固定在固相基质例如玻璃珠或烧瓶等上的抗体来分离造血干细胞或祖细胞。识别干细胞或祖细胞表面标记的抗体可以与其它化学基团融合或共轭,例如可以用固定在固相载体上的亲和素或链霉亲和素去除的生物素,或者用荧光激活细胞分选法(FACS)中的荧光染料等。较好的是利用免疫亲和柱来进行分离。免疫亲和柱可以是任意形式的,但通常都具有一个填充床层反应器。这类生物反应器内的填充床最好用均匀包裹了某种基质的多孔性材料制成。具有较高的表面积-体积比的多孔性材料可以使细胞混合物流经很大的接触面积而不阻碍床层外细胞的流动。典型的基质包括亲和素和链霉亲和素,但是也可以使用其它常用基质。基质必需因其本身的性质,或是通过增加某种化学基团而表现出对细胞结合蛋白例如单克隆抗体上的基团的高度亲和性。单克隆抗体能够识别要被分离的细胞上的表面抗原,而且通常经进一步修饰而具有生物素基团。众所周知的是,生物素具有对亲和素的高度亲和性,因而这些物质之间的亲和性可以去除固定到填充床的表面的单克隆抗体。这类柱是本领域众所周知的,参见Berenson等,《细胞生物化学杂志》(J.Cell.Biochem.)10D:239(1986)。该柱用PBS溶液洗涤以去除未结合的物质。可以利用常规方法从玻璃珠上释放靶细胞。例如,PCT公开WO93/08268中描述了以上所述的这类免疫亲和柱,即利用了固定在以亲和素包裹的填充床上的生物素化抗CD34单克隆抗体。对这种方法的一种改变是在分离方法中使用以可去除方式结合于固定表面的细胞结合蛋白,例如前文所述的单克隆抗体或flt3配体。然后令这些结合的细胞结合蛋白与集得的细胞混合物接触,培养足够的时间以分离所需的细胞。
或者,识别细胞表面抗原的单克隆抗体可以用荧光标记例如生色团或荧光团进行标记,然后利用根据标记产物有无或量进行的细胞分选进行分离。
而集得的CD34+细胞然仅与flt3配体接触,或者在与flt3配体接触的同时或其后与如下的一种或多种细胞因子联合一起接触:GM-CSF、TNF-α、IL-3、IL-4、c-kit-配体或GM-CSF/IL-3融合蛋白。然后令CD34+细胞分化并定型为树突状细胞系的细胞。然后收集该树突状细胞,从而可以(a)给患者使用以放大由免疫系统和T细胞或B细胞介导的对抗原的免疫应答,(b)该树突状细胞在给患者使用前与抗原接触,(c)用编码表面抗原特异性多肽的基因转染,或(d)在体外与抗原接触,然后加工并向采集自患者的T细胞提呈该抗原,然后给患者使用该抗原特异性T细胞。
更特殊的是,本发明使用有效量的flt3-配体在体内,例如在患者的外周血或其它组织或器官例如脾脏中,增加或集汇树突状细胞。通过增加患者树突状细胞的量,这些细胞本身可能可以被用于向T细胞提呈抗原。例如,抗原可能是已经存在于患者体内的,例如肿瘤抗原,或者是细菌或病毒抗原。所以,flt3-配体可以被用于加强患者对已经存在的抗原的由淋巴细胞(例如T细胞和B细胞介导)或髓样细胞介导的免疫应答,由此潜在地形成更为有效的向患者T细胞的抗原提呈。或者,flt3-配体可以在对患者给以某种抗原进行免疫之前、同时或之后给予。所以,作为疫苗佐剂,flt3-配体可以在体内产生大量树突状细胞,从而更有效地提呈抗原。整体应答是对抗原的更强、更好的免疫应答和更有效的免疫作用。
flt3-配体全身给药不仅是有效的疫苗佐剂,而且如前文所述,可以放大对已经存在的抗原的免疫应答。例如,本发明还证明,给患肿瘤的小鼠使用flt3-配体至少显著降低了肿瘤的生长速度,而且在很多小鼠中引起了肿瘤排斥反应。实施例3中给出了更为具体的数据。所以,flt3-配体是一种在体内对抗原产生有效免疫应答的重要的细胞因子。
由于具有产生树突状细胞的能力,flt3-配体还可以用于促进被移植的组织或器官的存活。当同种异体器官或其它组织被移植到宿主中时,它们能够自供体转移干细胞、不成熟树突状细胞和成熟树突状细胞。这些细胞被称为过客细胞(passenger cell),可以移植到宿主的造血系统中。此外,宿主的干细胞、不成熟树突状细胞或成熟树突状细胞可能向供体的器官或组织移植。由此可能因为来自宿主和供体组织的不成熟树突状细胞与“另一方”的T细胞之间发生反应而在移植物和宿主之间形成一种耐受性。这种反应可能包括树突状细胞表达的识别主要组织相容性复合体(MHC)的T细胞的缺失。由此,供体细胞被“筛选”,使得它们不能识别和反抗宿主(即不发生移植物抗宿主疾病),而宿主T细胞经筛选后不能识别和反抗移植物(即不发生移植物排斥)。所以,可以获得相互耐受性,由此提高了移植物的被接受性。在移植前对宿主或供体给予flt3-配体能够在这些宿主或供体内产生更多量的树突状细胞,从而提高移植物的耐受性和存活率。
为了培养树突状细胞,可以使用多种生长和培养培养基,本领域的一般技术人员可以方便地确定这类培养基的组成。合适的生长培养基是含有营养物或代谢添加剂的溶液,包含无血清的或基于血清的那些。生长培养基的实例RPMI,TC199,Iscoves改进的Dulbecco’s培养基(Iscoves等,《实验医学杂志》(F.J.Exp.Med.)147:923(1978)),DMEM,Fischer的α培养基,NCTC,F-10,Leibovitz’sL-15,MEM和McCoy培养基。对本领域技术人员来显而易见的具体的营养物包括血清白蛋白、转铁蛋白、类脂、胆固醇、2-巯基乙醇或一硫代甘油之类的还原剂、丙酮酸、丁酸和氢可的松2-半琥珀酸酯之类的糖皮质素。更具体地说,标准培养基包括能源、维生素或其它支持细胞生长的有机化合物,起稳定培养基pH作用的缓冲液例如HEPES、Tris,以及各种无机盐。具体可参见在此列为参考的PCT公开WO95/00632,其中描述了大量无血清细胞生长培养基。
对于本发明的任何一种体外方法来说,外周血祖细胞(PBPC)和外周血干细胞(PBSC)都是利用本领域已知的apheresis方法收集的。参见例如Bishop等,《血液》(Blood)第83卷,第2期,pp.610-616(1994)。简而言之,使用常用设备,例如Haemonetics V50型apheresis仪(Haemonetics,Braintree,MA)收集PBPC和PBSC。通常,每次采集4小时,每周不超过5次,直至收集到例如约6.5×l08单核细胞(MNC)/kg患者。将细胞悬浮在标准培养基中,然后离心去除红细胞和嗜中性白细胞。抽取位于两相之间界面的细胞(在本领域中又称buffy层)并悬浮在HBSS中。悬浮细胞大多数是单核的,细胞混合物中主要是早期干细胞。然后可将形成的干细胞悬浮液与生物素标记的抗CD34单克隆抗体或其它细胞结合手段接触。接触期要保持足够长的时间,以使抗CD34单克隆抗体和干细胞表面上的CD34抗原之间充分反应。通常,至少1小时。然后将细胞悬浮液与试剂盒中的分离装置接触。分离装置可包含用亲和素包裹的微珠填充的柱。这类柱是本领域所熟知的,参见Berenson等,《细胞生物化学杂志》(J.CellBiochem.)10D:239(1986)。该柱用PBS液洗涤以去除未结合的物质。利用常规方法可将靶干细胞从微珠和抗CD34单克隆抗体上释放。由此获得的干细胞可以在调速冷冻仪(例如Cryo-Med,Mt.Clemens,MI)中冷冻,然后保存在液氮蒸汽中。可以使用10%的二甲基亚砜作为冷冻保护剂。从供体的采样全部完成后,将干细胞解冻并集合。将含有干细胞、生长培养基、诸如McCoy 5A培养基,0.3%琼脂和以下扩增因子中的至少一种:重组人GM-CSF、IL-3、重组人flt3-配体和重组人GM-CSF/IL-3融合分子(PIXY321)浓度在约200μ/ml,在37℃、5%CO2、饱和湿度空气中培养14天。还可以在培养物中加人人IL-1α或IL-4。最好的扩增因子组合包含flt3-配体加上IL-3或GM-CSF/IL-3融合蛋白。
为了给人体内给药,flt3-配体可以按照已知用于制备药学上有用组合物的方法来配制。flt3-配体可以作为单独活性物质或与其它已知的活性物质一起与药学上合适的稀释剂(例如Tris-HCl、乙酸、磷酸)、防腐剂(例如硫柳汞、苯甲醇、对羟基甲酸[甲、丙]酯)、乳化剂、助溶剂、佐剂和/或载体组成混合物。《雷明顿药物科学》(Remington’s Pharmaceutical Science)第16版,Mack Publishing Co.中描述了合适的载体及其配方。此外,这类组合物还包含与聚乙二醇(PEG)复合的flt3-配体,金属离子或结合在以聚乙酸、聚乙醇酸、水凝胶为例的聚合物中,或结合在脂质体、微乳液、微胶粒、单层囊或多层囊(unilameller or multilamellarvesicles)、红细胞血影或球形未成熟细胞(spheroblasts)中的flt3-配体。这样的组合物将影响flt3-配体的物理状态、溶解性、稳定性、体内释放速度和体内清除速度。
flt3-配体可以局部、肠胃外或吸人给药。“肠胃外”包括皮下注射、静脉、肌肉、脑池内注射,或输注技术。这些组合物通常包含有效量的单独的flt3-配体或另与有效量的任何其它活性物质组合。这样的剂量和组合物中包含的期望的药物的浓度取决于许多因素,其中包括预期的用途、患者的体重和年龄、给药途径。可以根据动物试验来确定预备剂量,然后根据本领域认可的方法确定人用剂量大小。综上所述,flt3-配体的典型剂量在约每平方米10微克至约每平方米1000微克。较好的剂量范围在约每平方米1D0微克至约每平方米300微克。
除此之外,以下实施例说明了具体的实施方式,但并不限定本发明的范围。
实施例1
树突状细胞的产生
本实施例描述使用flt3-配体在体外产生大量树突状细胞。如前所述分离具有CD34+表型的细胞,例如,首先通过如前文所述产生buffy层细胞。然后将取自buffy层的细胞与CD34特异性抗体一起培养。然后将选择出的CD34+细胞培养在加有20ng/ml GM-CSF、IL-4和TNF-α或100ng/ml flt3-配体或c-kit配体的McCoy加强培养基中。在37℃,10%CO2,湿润空气中连续培养约2周。然后利用流式细胞仪(flow cytometry)分选表达CD1a+和HLA-DR+的细胞。GM-CSF、IL-4和TNF-α的组合将培养2周后获得的细胞数量提高了6至7倍。flt3-配体和c-kit配体的组合令绝对细胞数量增加了12至13倍。这与单用flt3-配体或c-kit配体的18倍扩增和合用flt3-配体和c-kit配体的达34倍扩增有关。对细胞的表型分析证明,在所有被检因子组合中,这些细胞中60至70%是HLA-DR+,CD86+,40至50%是表达CD1a的细胞。添加flt3-配体将CD1a+细胞的绝对数量提高了5倍。c-kit配体令这类细胞增加了6.7倍,flt3-配体和c-kit配体的组合提高了11倍。对生成的细胞的在MLR中的功能分析显示,flt3-配体或c-kit配体的存在在增加获得数量的同时并不影响生成的树突状细胞的刺激能力(stimulatory capacity)。
实施例2
flt3-配体在树突状细胞扩增中的用途
本实施例说明了一种利用flt3-配体扩增树突状细胞的方法。在收集细胞之前,可能需要集汇或增加循环的PBPC和PBSC量。集汇可以提高PBPC和PBSC收集,这是通过在收集细胞前给患者静脉内使用flt3-配体或sagramostim(Leukine,Immunex Corporation,Seattle,Washington)实现的。在给药flt3-配体之后或同时可以联合给以其它生长因子,例如CSF-1、GM-CSF、c-kit配体、G-CSF、EPO、IL-1、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、GM-CSF/IL-3融合蛋白、LIF、FGF及它们的组合。利用本领域已知的apheresis法收集集汇或非集汇的PBPC和PBSC。参见,例如Bishop等,《血液》第83卷,第2期,pp.610-616(1994)。简而言之,使用常用设备,例如Haemonetics V50型apheresis仪(Haemonetics,Braintree,MA)收集PBPC和PBSC。通常,每次采集4小时,每周不超过5次,直至收集到约6.5×108单核细胞(MNC)/kg患者。测定集得的PBPC和PBSC等份中的粒细胞-巨噬细胞集落形成单位(CFU-GM)含量,即通过用不含钙和镁的Hank平衡盐溶液(HBSS)约1∶6稀释,然后铺在淋巴细胞分离培养基(Organon Teknika,Durham,North Carolina)上。离心后,收集界面上的MNC,用HBSS洗涤和再悬浮。将含有约300,000MNC、改进McCoy 5A培养基、0.3%琼脂、200U/ml重组人GM-CSF、200U/ml重组人IL-3和200U/ml重组人G-CSF的1毫升的等份在37℃、5%CO2、饱和湿度的空气中培养14天。或者,在培养物中还可以加入flt3-配体或GM-CSF/IL-3融合分子(PIXY321)。这些培养物以Wright染料染色,用解剖显微镜计数CFU-GM集落(Ward等,《实验血液学》(Exp.Hematol.)16:358(1988))。或者,可以利用Siena等在《血液》第77卷,第2期,pp400-409(1991)中所述的CD34/CD33流式细胞仪法或其它本领域已知的方法分析CFU-GM集落。
含有CFU-GM的培养物在调速冷冻仪(例如Cryo-Med,Mt.Climens,MI)中冷冻,然后保存在液氮蒸汽中。10%的二甲基亚砜可作为冷冻保护剂。从患者的采样全部完成后,将含有CFU-GM的培养物解冻和汇合。解冻后的细胞采集物与flt3-配体单独接触,或者在其后或同时与其它前文所述的细胞因子联合物接触。与flt3-配体的这种接触将促使CFU-GM成为树突状细胞系。树突状细胞被通过静脉回输给病人。
实施例3
flt3-配体在放大抗肿瘤免疫应答中的用途
本实施例说明了利用flt3-配体在体内放大抗肿瘤免疫应答的方法。雌性C57BL/10J(B10)小鼠(Jackson实验室,Bar Harbor,ME)在腹部中线位置皮下注射总量为50微升的5×105能活的B10.2或B10.5纤维肉瘤肿瘤细胞。B10.2和B10.5纤维肉瘤细胞系源自B10细胞系,在现有技术中已被描述过,参见在此引为参考的Lynch等,《欧洲免疫学杂志》(Euro.J.Immunol.)21:1403(1991)。纤维肉瘤B10.2细胞系是通过皮下植入含5毫克甲基胆蒽的石蜡小丸来诱导的,B10.5细胞系是通过长期曝露于紫外照射来诱导的。肿瘤细胞系体外维持于含5%FBS、2nM L谷氨酸、50U/ml青霉素和50微克/毫升链霉素的α改进MEM中。在19天(除非另外说明)中,每日皮下注射总体积100微升的重组人flt3-L(10微克/注射)。对照组的小鼠相同地注射含100纳克MSA的相同体积的缓冲液。在以肿瘤攻击后,以肿瘤的大小对时间作图,由此确定肿瘤的生长速度。肿瘤的大小计算为测径器测得的两垂直直径之积,表达为特殊处理组中荷瘤的小鼠的平均肿瘤大小。在下述资料给出了实验结束时每个处理组中与受到攻击的数量相比,荷瘤的小鼠的数量。
表I中,数据采编自6次不同的实验,其中荷瘤的小鼠接受了flt3-配体或MSA处理。在50个接受flt3-配体处理的小鼠中有19个观察到肿瘤的完全消除,而在30个MSA处理小鼠中仅有1个(采用Fishers Exact Test的p<0.0001)。而且还证明了与MSA处理的小鼠(肿瘤攻击后第5周的;平均肿瘤大小为185+/-17mm2)相比,用flt3-配体处理的小鼠中的肿瘤生长速度显著减慢(肿瘤攻击后第5周的荷瘤鼠中平均肿瘤大小为60+/-8mm2)(采用Variance分析的p为0.0001)。
表I
6次实验的纤维肉瘤+/-flt3-L状况
肿瘤大小(mm2)
肿瘤攻击后的周数 | MSA对照(100纳克/天) | 标准误差 | flt3-L(10微克/天) | 标准误差 |
0 | 0 | 0 | 0 | 0 |
1 | 25 | 2.6 | 24 | 2.2 |
2 | 62 | 7.5 | 49 | 3.6 |
3 | 98 | 10.6 | 49 | 3.9 |
4 | 149 | 14.5 | 50 | 5 |
5 | 185 | 16.8 | 60 | 8.4 |
与对照相比,使用flt3-配体显著抑制了肿瘤的大小。所以,以上数据表明,在放大对外源抗原,尤其是癌症的免疫应答中,flt-3配体是一种重要的细胞因子。
Claims (4)
1.一种在体外使造血干细胞或祖细胞向树突状细胞趋动的方法,它包括令所述的造血干细胞或祖细胞与能够产生树突状细胞群的足量flt3-配体接触。
2.一种体外制备表达抗原的纯化树突状细胞群的方法,它包括:
(a)令造血干细胞或祖细胞与能够产生树突状细胞群的足量flt3-配体接触;
(b)或者(i)令树突状细胞与抗原特异性肽接触,或者(ii)用编码抗原特异性肽的基因转染树突状细胞;
(c)让树突状细胞加工和表达该抗原;
(d)纯化表达该抗原的树突状细胞。
3.根据权利要求2所述的方法,其中的步骤(a)还包括令造血干细胞或祖细胞与选自以下组的一种分子接触;GM-CSF、IL-4、TNF-α、IL-3、c-kit配体以及GM-CSF和IL-3的融合体。
4.一种体外制备抗原特异性T细胞的方法,它包括:
(a)令造血干细胞或祖细胞与能够产生树突状细胞群的足量flt3-配体接触;
(b)或者(i)令树突状细胞与抗原特异性肽接触,或者(ii)用编码抗原特异性肽的基因转染树突状细胞;
(c)让树突状细胞加工和表达该抗原;
(d)让树突状细胞向T细胞提呈该抗原。
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1996
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DE69637942D1 (de) | 2009-07-09 |
KR100514957B1 (ko) | 2005-11-25 |
CA2232865A1 (en) | 1997-04-10 |
ATE432085T1 (de) | 2009-06-15 |
CZ97798A3 (cs) | 1999-02-17 |
NO981374D0 (no) | 1998-03-26 |
KR100676792B1 (ko) | 2007-02-02 |
EP0871487B1 (en) | 2009-05-27 |
KR19990063818A (ko) | 1999-07-26 |
EP0871487A4 (en) | 2004-09-01 |
WO1997012633A1 (en) | 1997-04-10 |
EA199800272A1 (ru) | 1998-10-29 |
EP0871487A1 (en) | 1998-10-21 |
RO120579B1 (ro) | 2006-04-28 |
BR9610802A (pt) | 1999-07-13 |
AU7392296A (en) | 1997-04-28 |
IS4703A (is) | 1998-03-26 |
KR20040079421A (ko) | 2004-09-14 |
EE9800105A (et) | 1998-10-15 |
NZ321039A (en) | 2001-03-30 |
NO981374L (no) | 1998-06-03 |
CN1229359A (zh) | 1999-09-22 |
JP3631496B2 (ja) | 2005-03-23 |
PL325964A1 (en) | 1998-08-17 |
TR199800607T1 (xx) | 1998-06-22 |
AU697539B2 (en) | 1998-10-08 |
SI9620116A (sl) | 1999-08-31 |
SK40898A3 (en) | 1999-04-13 |
LV12085B (en) | 1998-09-20 |
LV12085A (lv) | 1998-07-20 |
JPH11513389A (ja) | 1999-11-16 |
PL187329B1 (pl) | 2004-06-30 |
ES2323818T3 (es) | 2009-07-24 |
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