CN1167440A - 抗癌免疫疗法的应激蛋白-肽复合物 - Google Patents
抗癌免疫疗法的应激蛋白-肽复合物 Download PDFInfo
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Abstract
本发明公开的是一种抑制哺乳动物体内肿瘤增殖的方法。该方法包括以下步骤:(a)从预先从哺乳动物中得到的肿瘤细胞中分离应激蛋白-肽复合物,(b)将分离的应激蛋白-肽复合物回输给哺乳动物,以刺激哺乳动物对分离出该复合物的肿瘤的免疫应答。在本发明实践中具有特殊应用的应激蛋白-肽复合物包括Hsp70-肽、Hsp90-肽和gp-96肽复合物。
Description
发明领域
本申请总体上涉及癌症治疗领域,具体地说,涉及人癌的免疫疗法。
发明背景
人们已经发现,预先免疫近交繁殖的小鼠和大鼠,可以抵抗由同一遗传背景的小鼠和大鼠而来的肿瘤。(Gross(1943)癌症研究3:323-326;Prehn等(1957)国家癌症学会杂志18:769-778;Klein等(1960)癌症研究20:1561-1572;Old等(1962)纽约科学院年刊101:80-106;综述见Srivastava等(1988)现代免疫学9:78-83)。这些研究不仅指出接种没有活性的癌细胞的小鼠可以对后续的活性癌细胞的攻击产生免疫力,并且表明肿瘤特异性抗原的存在。
进一步的研究揭示了这种预防性诱导免疫力的现象具有肿瘤特异性。尽管小鼠能对用来免疫它们的瘤细胞产生特异的免疫力,它们对于其它无关的肿瘤的攻击仍然敏感。(Basombrio(1970)癌症研究30:2458-2462,Globerson等(1964)国家癌症学会杂志32:1229-1243)。癌细胞免疫原性的证实引起了对能够引发对肿瘤攻击产生抗性的癌源分子的研究。一般的途径是分级分离癌细胞来源的蛋白,然后分别检测它们使小鼠对该级分由之制备的癌产生免疫的能力(Srivastava(1988)同上;Old(1981)癌症研究41:361-375)。利用这种方法已鉴定出一些蛋白,但是,这些蛋白中大部分与一类被称作应激诱导蛋白或应激蛋白的蛋白相关(Lindquist等(1988)遗传学综述年刊22:631-677)。由于应激蛋白在性质上是最高度保守和最丰富的蛋白之一,因此,看来它们并不是肿瘤特异性抗原的候选物。随后发现应激蛋白以非共价形式与多种肽类结合,形成应激蛋白--肽复合物。(Gething等(1992)自然355:33-45;Lindquist等(1988)同上;Young(1990)免疫学综述年刊8:401-420;Flynn等(1991)自然353:726-730)。
研究显示,在用ATP进行处理时应激蛋白--肽复合物会失去其免疫原性。(Udono等(1993)实验医学杂志178:1391-1396)。这种处理能把应激蛋白-肽复合物解离成应激蛋白和肽两部分。考虑到从正常细胞和肿瘤细胞得到的应激蛋白的结构没有差别,并且应激蛋白以ATP依赖方式与多种肽类相结合,这种应激蛋白-肽复合物的抗原性似乎并不是应激蛋白本身形成的,而是由与应激蛋白结合的肽形成的。
因此,本发明的一个目的是为治疗性地抑制哺乳动物肿瘤的增殖提供一种新的方法。这里所描述的方法不要求对特异性抗原决定簇进行分离和表征,并且,可以为制备和利用对抑制哺乳动物特异性的预定肿瘤增殖有效的免疫原性组合物提供一种更快的方法。
本发明的这一目的和其他的目的及特征在下面的说明和权利要求中是显而易见的。
发明概述
应激蛋白与分离出它们的细胞的抗原性肽相伴随这一发现,为容易地分离预选肿瘤的抗原性肽提供了一种方法。一旦分离完毕,这种应激蛋白-肽复合物又被回输给分离出它的动物,以引发对原有肿瘤的免疫应答。因此,这种方法不必对肿瘤特异性抗原进行分离和表征,可以使技术人员容易地制备出对抗预选肿瘤的有效的免疫原性组合物。
广义地讲,本发明为抑制哺乳动物中预选肿瘤的增殖提供了一种方法。这种方法包括给治疗中的哺乳动物施用一种组合物,该组合物包含与应激蛋白-肽复合物联合应用的药用载体。这种复合物是从预先从哺乳动物切下的肿瘤细胞中得到的,其特征为可以有效地启动哺乳动物对分离出该复合物的肿瘤细胞的免疫应答。随后将这种复合物以足够的数量回输给哺乳动物,以引发哺乳动物对肿瘤细胞的免疫应答,由此,可以抑制哺乳动物中任何仍然残存的肿瘤细胞的增殖。
预计这种方法可以和其它常规的癌症治疗方法(包括,例如,外科手术、放疗、化疗)联合应用。例如,技术人员可以在手术切除癌组织之后,用本文描述的方法,从切除的组织中分离应激蛋白-肽复合物并把这种复合物回输给哺乳动物。这种复合物可随后诱导机体对在外科手术中没有被切除的仍然存在的肿瘤细胞产生特异性免疫应答。当原发肿瘤转移到身体的不同部位时,这种方法对癌症治疗是适合的。
本文所用的术语“肿瘤”,可以理解为细胞的任何一种异常或不可控制的生长,这种生长可导致对正常组织的侵害。可以预计这一术语也包括已转移了的异常生长或生长失控的细胞,即已经从身体原发部位(即原发肿瘤)扩散到空间上与原发部位不同的继发部位的异常细胞。
本文所用的术语“应激蛋白”,可以理解为任何一种满足下列条件的细胞蛋白:当细胞受到应激性刺激时,它在细胞内的浓度升高,它能够结合其它的蛋白或肽,在ATP存在或pH值低的情况下能够释放结合的蛋白或肽。应激性刺激包括但不仅限于热休克、营养缺乏、代谢紊乱、氧基和细胞内病原体的感染。
第一种要鉴定的应激蛋白是热休克蛋白(Hsp)。正如它的名字所暗示的,Hsp典型地是由对热休克产生应答的细胞诱导产生的。目前已经鉴定出的哺乳动物三个主要Hsp家族,包括Hsp60、Hsp70和Hsp90。数字反映了应激蛋白以千道尔顿为单位的近似分子量。每个家族的成员都高度保守,见例如,Bardwell等(1984)美国国家科学院院刊81:848-852;Hickey等,(1989)分子细胞生物学9:2615-2626;Jindal(1989)分子细胞生物学9:2279-2283,其公开内容在此引入作为参考。到目前为止,鉴定出的哺乳动物Hsp90家族成员包括胞液Hsp90(亦称作Hsp83)和内质网对应物Hsp90(亦称作Hsp83)、Hsp87、Grp94(亦称作ERp99)和gp96。见例如(Gething等(1992)自然355:33-45,其公开内容在此引入作为参考。到目前为止,鉴定出的Hsp70家族成员包括胞液Hsp70(亦称作p73)和Hsc70(亦称作p72);内质网对应物Bip(亦称作Grp78);线粒体对应物Hsp70(亦称作Grp75),Gething等(1992)同上。到目前为止,哺乳动物Hsp60的家族成员仅仅在线粒体中鉴定出。Getnhing等(1992)同上。
此外,已经发现Hsp60、Hsp70和Hsp90家族由在氨基酸序列上与应激蛋白相关(例如有大于35%的氨基酸序列一致)的蛋白组成,但是他们的表达水平并不因应激性刺激而改变。因此,可以预计在这里所用的应激蛋白的定义,包括其他蛋白,其突变蛋白,类似物和变异体,与这三个家族的成员最少具有35%-55%,优选55%-75%,最优选75%-85%的氨基酸序列一致性,这三个家族的成员在细胞内的表达水平在对应激性刺激应答时受到激发。
这里所用的术语“肽”,可理解为任何一种从哺乳动物肿瘤细胞分离到的以应激蛋白-肽复合物形式存在的氨基酸序列。
这里所用的术语“免疫原性应激蛋白-肽复合物”,可以理解为任何一种能够从哺乳动物肿瘤细胞分离并且包含与肽非共价结合的应激蛋白的复合物。这种复合物又以有效地引发哺乳动物对分离出它的肿瘤细胞的免疫应答为特征。
这里所用的术语“免疫应答”,可以理解为哺乳动物在抗原刺激下产生的任何一种目的为从哺乳动物中清除抗原的细胞过程。这种免疫应答典型地由一种或多种性质上属于淋巴细胞和/或吞噬细胞的细胞群所介导。
在本发明更特殊的方面中,应激蛋白-肽复合物中的应激蛋白选自Hsp70、Hsp90和gp96。包含有Hsp70-肽、Hsp90-肽和gp96-肽复合物的应激蛋白-肽复合物可同时从一批由哺乳动物切割而来的肿瘤细胞中分离得到。可以预计,在免疫治疗的过程中可把一种或多种前面提到的复合物施用给哺乳动物,以刺激机体对肿瘤产生最合适的免疫应答。
预计,本文所描述的方法对人癌的治疗特别有效。但是,可以预计,本文所描述的方法也对其它哺乳动物癌症的免疫疗法有效,这些动物例如农场动物(即牛、马、山羊、绵羊和猪)和家庭宠物(即猫和狗)。
在本发明的另一方面中,预计免疫应答受T细胞级联方式的影响,更具体地说,是受细胞毒T细胞级联方式的影响。这里所用的术语“细胞毒T细胞”,可理解为任何一种表达细胞表面糖蛋白标志CD8的T淋巴细胞,这种淋巴细胞能靶向和裂解细胞表面带有I型组织相容性复合物的被细胞内病原体感染的靶细胞。
在本发明的另一方面中,这种应激蛋白-肽复合物可以和一些治疗有效量的细胞因子联合施用给哺乳动物。这里所用的术语“细胞因子”指任何一种影响其它介导免疫应答细胞的功能的分泌型多聚肽。因此,可以预计这种复合物可以和细胞因子一起施用以增强针对肿瘤的免疫应答。优选的细胞因子包括,但不仅限于,白细胞介素-1α(IL-1α)、白细胞介素-1β(IL-1β)、白细胞介素-2(IL-2)、白细胞介素-3(IL-3)、白细胞介素-4(IL-4)、白细胞介素-5(IL-5)、白细胞介素-6(IL-6)、白细胞介素-7(IL-7)、白细胞介素-8(IL-8)、白细胞介素-9(IL-9)、白细胞介素-10(IL-10)、白细胞介素-11(IL-11)、白细胞介素-12(IL-12)、干扰素α(IFN-α)、干扰素β(IFN-β)、干扰素γ(IFN-γ)、肿瘤坏死因子α(TNFα)、肿瘤坏死因子β(TNFβ)、粒细胞集落刺激因子(G-CSF)、粒细胞/巨噬细胞集落刺激因子(GM-CSF)、和转化生长因子β(TGF-β)。
该复合物可利用本领域众所周知的技术与常规的药用载体、佐剂或赋形剂相结合。应激蛋白-肽复合物家族必要的施用剂量和方式依赖于不同的因素,比如生理条件下复合物的稳定性、复合物在诱导免疫应答时的有效性、肿瘤的大小及分布、受治疗哺乳动物的年龄、性别和重量。
典型地,施用该复合物的量应该足以诱发哺乳动物对分离出该复合物的肿瘤产生免疫应答和抑制肿瘤细胞的增殖。应激蛋白-肽复合物的施用量的范围优选的是1-1000微克复合体/公斤哺乳动物体重/次施用,最优选的是大约100-250微克复合体/公斤哺乳动物体重/次施用。预计一个大约75公斤体重的人的典型剂量范围是大约5-20mg。此外,预计通过反复向个体施用这种复合物可以提高机体的免疫应答强度。哺乳动物优选地至少每隔一周接受两剂应激蛋白-肽复合物。如果必要的话,可通过稍后施用这种复合物而在较晚的时间增强免疫应答。预计,本领域的技术人员通过常规的实验可找到最适的剂量和免疫方案。
发明详述
本发明基于如下观察,即应激蛋白-肽复合物与分离出它们的细胞的抗原性肽相伴随。常规的癌症治疗基于对肿瘤特异性抗原进行分离、表征,然后以此抗原作为特定治疗方案的目标。由于哺乳动物癌症的抗原差异性,所以,对每种特定肿瘤的特定肿瘤抗原进行分离和表征是不现实的。本发明避免了对每种被治疗的肿瘤的肿瘤特异性抗原进行分离和表征,为癌症的免疫治疗提供了另外一种途径。
本发明为抑制哺乳动物预选肿瘤的增殖提供了一种方法。这种方法包括从接受治疗的哺乳动物分离或获得肿瘤细胞。可通过采用本领域熟知的外科手术方法容易地完成。典型地,肿瘤细胞可从进行常规肿瘤外科切除的哺乳动物中获得。此方法随后涉及包括从切除的肿瘤细胞中分离应激蛋白-肽复合物。这可以利用下面详细描述的任何一种分离方案完成。这种应激蛋白-肽复合物的特征在于,当它们又回输给哺乳动物时,能起始机体对分离出它们的同一类型肿瘤细胞产生特异性免疫应答。最后,这种方法包括把应激蛋白-肽复合物回输给哺乳动物,所用的量足以在哺乳动物中诱发机体对肿瘤细胞产生免疫应答,由此抑制哺乳动物中残存的任何肿瘤细胞的增殖。
预计这种方法可以和一种或多种常规的癌症治疗方法(包括例如外科手术、放疗和化疗)联合应用。例如,技术人员可用这里所描述的方法在肿瘤组织切除以后,从中分离应激蛋白-肽复合物,并把它回输给该哺乳动物。于是,该复合物可诱导哺乳动物对手术中没有去除的肿瘤细胞产生特异性免疫应答。另外,本文所描述的方法为原发肿瘤已转移到机体多个部位时的癌症治疗提供了一种新的方法。例如,在癌症已转移,不能实行外科手术的情况下,可单独应用这种应激蛋白-肽复合物或与另一种癌症治疗中标准的化疗药剂联合应用。
预计,本发明在人癌的免疫治疗方面有特别的应用,但是,这里所描述的方法也适于例如农场动物(即牛、马、绵羊、山羊和猪)和家庭宠物(即猫和狗)的癌症治疗中。
本方法相对于常规方法的主要优点在于不必对每一种肿瘤的肿瘤特异性抗原进行分离、表征。一旦分离出应激蛋白-肽复合物,就可不用进一步表征、而简单地回输给哺乳动物。由于分离免疫原性复合物的方法是本领域常规而已知的,所以技术人员可快速地、常规地为每个接受治疗的个体制备出“定做的”特异免疫原性组合物。
这种方法优于常规方法的另一优点在于把这种纯化的应激蛋白-肽复合物回输给分离出它们的个体,可以消除给治疗中的哺乳动物注射潜在转化剂(即转化DNA)和/或免疫抑制剂的危险,当复合物存在于生化性质尚未明确的肿瘤或肿瘤提取物中时,它们的应用就会成为问题。此外,应激蛋白-肽复合物在无佐剂时就可诱导明显的肿瘤免疫。因此,尽管佐剂能进一步提高复合物的免疫治疗特性,但是它们的存在在诱导明显的免疫应答时并不是先决条件。
预计,这种方法可用于不同肿瘤的治疗,例如,间质组织来源的肿瘤(肉瘤)即,纤维肉瘤;黏液肉瘤;脂肪肉瘤;软骨肉瘤;成骨肉瘤;血管肉瘤;内皮肉瘤;淋巴管肉瘤;滑膜肉瘤;间皮肉瘤;Ewing’s瘤;骨髓白血病;单核细胞白血病;恶性淋巴瘤;淋巴细胞白血病;浆细胞瘤;平滑肌肉瘤和横纹肌肉瘤。
此外,预计这种方法可用于上皮组织来源的肿瘤(癌)的治疗即,鳞状细胞或表皮癌;基细胞癌;汗腺癌;皮脂腺癌;腺癌;乳状癌;乳腺癌;囊腺癌;骨髓癌;未分化癌(单成份癌);支气管癌;鳃裂癌;黑色素癌;肾细胞癌;肝细胞癌;胆管癌;乳头状癌;过渡型细胞癌;鳞状细胞癌;绒毛膜癌;精原细胞瘤;胚胎癌;恶性畸胎瘤和畸胎癌。下面将详细论述制备这种有效地诱导对这些肿瘤产生免疫应答的组合物的一般方法。
尽管不希望被理论所束缚,预计这种应激蛋白-肽复合物通过T细胞级联刺激机体对分离出它们的肿瘤细胞产生免疫应答。先前的实验证明,用始发于同系大鼠或小鼠的肿瘤分离出的应激蛋白-肽复合物预先免疫小鼠,小鼠可针对分离出这种复合物的肿瘤产生免疫性抵抗。但是,不能针对抗原性不同的肿瘤产生免疫力。而且,从正常组织中分离到的应激蛋白-肽复合物不能诱导小鼠对任何受试肿瘤的抵抗。见例如,Srivastava等(1984)国际癌症杂志33:417;Srivastava等(1986)美国国家科学院院刊83:3407;Palladino等(1987)癌症研究47:5074;Feldweg等(1993)细胞生化杂志.增刊17D:108(摘要);Udono(1993)细胞生化杂志.增刊17D:113;Udono(1993)实验医学杂志178:1391-1396。这些内容在此引入作为参考。最近已经确定预防免疫以T细胞级联方式被介导,更具体地说,由细胞毒T细胞级联介导。见例如,Blachere等(1993)免疫疗法杂志14:352-356,这些内容在此引入作为参考。由此,可预计应激蛋白-肽复合物也许由同样的机制介导它们有效的治疗作用;具体地说,通过细胞毒T细胞级联。
预计,这种应激蛋白-肽复合物典型地可以从治疗中哺乳动物切除的肿瘤组织中直接分离。但是,在某些情况下,用于分离复合物的肿瘤组织的数量有限,因此,预计在分离应激蛋白-肽复合物之前,可通过运用本领域众所周知的技术使切除的肿瘤组织增殖。例如,切除的肿瘤组织可在体内增殖,例如,将肿瘤组织的标本转染给裸鼠,或在体外,例如可对培养中的肿瘤组织进行连续传代,随后,可收获这些增殖的瘤组织,并作为分离应激蛋白-肽复合物的初始物。
在本发明实践中有效的应激蛋白可定义为任何一种满足下列条件的细胞蛋白:当细胞受到应激性刺激时,该蛋白的细胞内的浓度升高;可以结合其它的蛋白或肽;在ATP存在或pH值低时,可释放结合的蛋白或肽。
第一种要鉴定的应激蛋白为Hsp,这是在对热休克产生应答的细胞内合成的。目前为止,已鉴定出哺乳动物Hsp的三个主要家族,包括Hsp60、Hsp70和Hsp90,这里的数字反映了应激蛋白以千道尔顿为单位的分子量。以后又发现在对其它应激性刺激,包括但不仅限于,营养缺乏、代谢紊乱、氧基和细胞内病原体的感染等产生应答时也可诱导出这些家族的许多成员。见例如:Welch(1993年5月)科学美国人56-64;Young(1990)同上;Craig(1993)科学260:1902-1903;Gething等,同上;Lindquist等(1988)同上,这些内容在此引入作为参考。预计,所有属于这三个家族的哺乳动物应激蛋白在本发明的实践中是有效的。
主要的应激蛋白在应激的细胞中积累到非常高的水平,在没有受到应激的细胞里则低到中等水平。例如,具有高度可诱导性的哺乳动物的Hsp70在常温下难以检测,但在受到热休克的细胞中则成为最活跃地合成的蛋白之一。(Welch等(1985)细胞生物杂志101:1198-1211)。不同的是,大部分,但不是全部哺乳动物细胞的Hsp90、Hsp60蛋白在常温下含量丰富,可由热进一步诱导。(Lai等(1984)分子细胞生物学4:2802-10;van Bergen en Henegouwen等(1987),基因进展1:525-31)。
到目前为止,已鉴定出的哺乳动物Hsp90家族成员包括胞液中的Hsp90(亦称作Hsp83)和内质网对应物Hsp90(亦称作Hsp83),Hsp87,Grp94(亦称作ERp99)和gp96(Gething等(1992)同上)。这些内容在此引入作为参考。鉴定出的Hsp70家族成员包括胞液Hsp70(亦称作p73)和Hsc70(亦称作p72);内质网对应物Bip(亦称作Grp78);线粒体对应物Hsp70(亦称作Grp75),Gething等(1992)同上。目前,哺乳动物Hsp60的家族成员仅仅在线粒体中鉴定出,Gething等(1992)同上。
应激蛋白是现存的最高度保守的蛋白之一。例如,DnaK,E.coli的Hsp70有大约50%的氨基酸序列与真核细胞的Hsp70蛋白一致。(Bardwell等,美国国家科学院院刊(1984)81:848-852)。相似地,Hsp60和Hsp90家族均具有高度的家族内保守性(Hickey等(1989)分子细胞生物学9:2615-2626;Jindal分子细胞生物学9:2279-2283)。此外,已发现Hsp60、Hsp70、Hsp90的家族由在序列上与应激蛋白相关的蛋白组成,例如,有大于35%的氨基酸序列一致,但是在应激状态下其表达水平并不改变。因此,预计这里所用的应激蛋白的定义包括其它的蛋白,其突变的蛋白、类似物和变异体,这些蛋白的氨基酸序列至少有35%-55%,优选55%-75%,最优选75%-85%与这三个家族成员的一致,在对应激性刺激应答的过程中,这三个家族成员的细胞内表达水平增高。
本发明的免疫原性应激蛋白-肽复合物包括任何一种含有以非共价形式结合的应激蛋白和肽的复合物,所述肽能在哺乳动物体内诱导免疫应答。优选的复合物包括但不仅限于,Hsp70-肽、Hsp90-肽和gp96-肽复合物。例如,在本发明的实践中,可应用哺乳动物的应激蛋白gp96,它是胞液Hsp90的内质网相对物。
以下将详细论述分离本发明实践中所用的应激蛋白-肽复合物的典型方法。
Hsp70-肽复合物的纯化
以前已经描述过Hsp70-肽复合物的纯化方法,见例如,Udono等(1993)同上。
首先,将肿瘤细胞悬浮在3体积的由5mM磷酸钠缓冲液(PH7)、150mM NaCl,2mM CaCl2,2mM MgCl2和1mM苯甲磺酰氟(PMSF)组成的1×裂解缓冲液中。接着,在冰浴中对细胞团进行超声处理,直到显微镜下检测99%以上的细胞裂解。作为超声处理的替代方法,可用机械剪切对细胞进行裂解,在这种方法中,细胞常重悬于30mM碳酸氢钠pH7.5、1mM PMSF的溶液中,冰浴中培养20分钟,然后在dounce匀浆器中匀浆,直到95%以上的细胞裂解。
然后,将裂解物离心1000g×10min,以去除未破裂的细胞、核和其它细胞碎片。得到的上清液再离心100,000g×90min,收集上清,与被含有2mM Ca2+和2mM Mg2+的磷酸盐缓冲液(PBS)平衡过的ConA琼脂糖混合。当细胞被机械剪切裂解时,可先用等体积的2×裂解缓冲液稀释,然后再与ConA琼脂糖混合。上清液可在4℃和ConA琼脂糖混合2-3h,收集未结合的物质,用10mM Tris乙酸盐pH7.5、0.1mM EDTA、10mM NaCl、1mM PMSF透析36小时(三次,每次100体积)。然后,将透析液离心17000rpm×20min(Sorvall SS34转头)。所得的上清液被收集,上Mono Q FPLC柱,该柱用20mM Tris乙酸盐pH7.5、20mMNaCl、0.1mM EDTA和15mM 2-巯基乙醇平衡液平衡。接着用20mM-500mM的NaCl梯度进行展开,洗脱级分由十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,使用合适的抗Hsp70抗体,用免疫印迹法进行表征。
合并与抗Hsp70抗体进行强烈免疫反应的级分,用硫酸铵沉淀Hsp70-肽复合物,具体地说,用50%-70%的硫酸铵分离。生成的沉淀通过17000rpm(SS Sorvall转头)离心获得,并用70%的硫酸铵洗涤。溶解洗涤的沉淀物,任何残存的硫酸铵可通过交联葡聚糖RG25(Pharmacia)柱进行凝胶过滤而被清除。
用这种方法能把Hsp70-肽复合物纯化得很均一。典型地,从1克细胞或组织能纯化到1mg Hsp70-肽复合物。
Hsp90-肽复合物的纯化
首先,将肿瘤细胞悬浮在3体积的由5mM磷酸钠缓冲液(PH7)、150mM NaCl、2mM CaCl2、2mM Mgcl2和1mM苯甲磺酰氟(PMSF)组成的1×裂解缓冲液中。接着,在冰浴中对细胞团进行超声处理,直到显微镜下检测99%以上的细胞裂解。作为超声处理的替代方法,可用机械剪切对细胞进行裂解,在这种方法中,细胞常重悬于30mM碳酸氢钠pH7.5、1mM PMSF的溶液中,冰浴中培养20分钟,然后在dounce匀浆器中匀浆,直到95%以上的细胞裂解。
然后,将裂解物离心1000g×10min,以去除未破裂的细胞、核和其它细胞碎片。得到的上清液再离心100,000g×90min,收集上清,与被1含有2mM Ca2+和2mM Mg2+的磷酸盐缓冲液(PBS)平衡过的ConA琼脂糖混合。当细胞被机械剪切裂解时,可先用等体积的2x裂解缓冲液稀释,然后再与ConA琼脂糖混合。上清液可在4℃下和ConA琼脂糖混合2-3小时,收集未结合的物质,用10mM Tris乙酸盐pH7.5、0.1mMEDTA、10mM NaCl、1mM PMSF透析36小时(三次,每次100体积)。然后,将渗析液离心17000rpm×20min(Sorvall SS34转头)。所得的上清液被收集,上Mono Q FPLC柱,该柱用裂解缓冲液平衡。用200mM-600mM的NaCl梯度洗脱蛋白。
洗脱的级分由SDS-PAGE分离,使用象3G3(Affinity Bioreagent)这样的抗Hsp90抗体,用免疫印迹法对含有Hsp90-肽复合物的级分进行鉴定。用这种方法能把Hsp90-肽复合物纯化得很均一。典型地,从1克细胞或组织能纯化到150-200μg Hsp90-肽复合物。
gp96-肽复合物的纯化
首先,将肿瘤细胞悬浮在3体积的由5mM磷酸钠缓冲液(PH7)、150mM NaCl、2mM CaCl2、2mM MgCl2和1mM苯甲磺酰氟(PMSF)组成的1×裂解缓冲液中。接着,在冰浴中对细胞团进行超声处理,直到显微镜下检测99%以上的细胞裂解。作为超声处理的替代方法,可用机械剪切对细胞进行裂解,在这种方法中,细胞常重悬于30mM碳酸氢钠pH 7.5、1mM PMSF的溶液中,冰浴中培养20分钟,然后在dounce匀浆器中匀浆,直到95%以上的细胞裂解。
然后,将裂解物离心1000g×10min,以去除未破裂的细胞、核和其它细胞碎片。将得到的上清液再离心100,000g×90min,收集上清,与被含有2mM Ca2+和2mM Mg2+的磷酸盐缓冲液(PBS)平衡过的ConA琼脂糖混合。当细胞被机械剪切裂解时,可先用等体积的2x裂解缓冲液稀释,然后再与ConA琼脂糖混合。上清液可在4℃下和ConA琼脂糖混合2-3小时。将浆液上柱,用1×裂解缓冲液洗涤,直到OD280降到基线。用1/2柱床体积的10%α-甲基甘露糖苷(α-MM)洗柱,用薄膜封柱,在37℃温育15分。接着把柱冷却到室温,从柱底移去薄膜,用5倍柱体积的α-MM洗柱。然后分离洗提物,用SDS-PAGE表征。通常,生成的gp96-肽复合物的纯度依细胞类型和组织对裂解液的裂解率不同而大约为60%-95%。
如果需要进一步纯化,可把样品加到用含有5mM磷酸盐的pH7缓冲液平衡的Mono Q FPLC柱。用0-1M的NaCl梯度从柱上洗脱蛋白。gp96级分的洗脱物在400-500mM NaCl之间。
作为可选方案,从100,000g细胞团分离来的gp96级分,可重悬于5体积含有1%脱氧胆酸钠(无钙离子和镁离子)的PBS液中,在冰浴中培养1小时,将所得上清液离心20,000g×30min,收集所得的上清液,在更换几次的PBS液(无钙离子和镁离子)中透析,以洗去去污剂。所得透析液离心100,000g×90min,将上清液进一步纯化。然后把钙和镁加到上清液,终浓度为2mM。接着把样品加到用含有5mM磷酸盐的pH7缓冲液平衡的Mono Q FPLC柱。用0-1M的NaCl梯度从柱上洗脱蛋白。gp96级分的洗脱物在400-550mM NaCl之间。
用这种方法能把gp96-肽复合物纯化得很均一。典型地,从1克细胞或组织能纯化到10-20μg gp96-肽复合物。
复合物的制剂和施用
一旦将应激蛋白-肽复合物从切除的肿瘤中纯化出来,它们就可回输给接受治疗的哺乳动物中,以刺激哺乳动物产生对分离出它们的肿瘤细胞的免疫应答。通过与生理可接受载体、赋形剂和稳定剂混合可对本发明的应激蛋白-肽复合物进行储存或准备施用。这些物质在所用剂量和浓度范围内应对接受者无毒性。
当这种复合物是水溶性时,它可在合适的缓冲液中配制,例如,PBS(5mM磷酸盐、150mM NaCl,pH7.1)或其它生理可接受溶液。可选地,如果所得复合物在水溶液中的溶解度低,可用象吐温、聚乙二醇这样的非离子表面活性剂配制。
口服或胃肠外施用时所用的溶液可通过任何一种药物学领域熟知的方法制备,例如,在Remington氏药物科学(Gennaro,A.,ed.),Mack出版社,1990中所描述的。这些制剂包括,例如,象聚乙二醇这样的聚亚烷基二醇、植物油、氢化萘等。直接应用的制剂,具体地说,可包括甘油和其它高粘度的组合物。生物相容的,优选生物可再吸收的聚合物,包括例如,透明质酸、胶原、磷酸三钙、聚丁酸酯、聚交酯、聚乙交酯和交酯/乙交酯的共聚物,它们也许是有效控制应激蛋白-肽复合物在体内释放的赋形剂。
用于吸入的制剂可包含赋形剂,例如,乳糖。水溶液可包含,例如,聚氧乙烯-9-十二烷基醚、甘胆酸盐和脱氧胆酸盐。油性溶液在滴鼻剂中也许有用。凝胶可以应用于鼻内局部施用。
通过与药用非毒性赋形剂和载体混合,本发明提供的复合物可配制成药物组合物。此外,制剂可优选地包含一种或多种佐剂。优选的佐剂包括但不限于,环氧乙烷-环氧丙烷的3嵌段共聚物、胞壁酰二肽和它的衍生物、减毒内毒素、皂角苷和它的衍生物如QS-21和脂质体。本发明进一步设想缓释剂型,在这种剂型中复合物可在一段延长的时间内释放。
按照本发明制备的应激蛋白-肽复合物家族的施用方式必须依赖于生理条件下复合物的稳定性和接受治疗的哺乳动物中的肿瘤的大小和分布。施用的复合物的优选剂量似乎也依赖于肿瘤的大小和分布、接受者的年龄、性别和体重、特定接受者的整体健康状况、复合物的相对生物效能、复合物的剂型、制剂中赋形剂的存在及其类型和施用的途径等不同因素。
通常,本发明的化合物可溶于水性生理缓冲液中,溶液中含有大约0.001-0.1%(w/v)的化合物,用于胃肠外施用。优选的剂量范围是大约1-1000微克复合物/公斤接受者体重/次施用,最优选的施用范围是大约100-250微克复合物/公斤接受者体重/次施用。具体地说,一个大约75公斤体重的人的典型剂量范围是5-20mg。但是,这些量要依照与复合物一起施用的佐剂而变化。
这种复合物优选地包含用标准方法可以施用的部分水溶液,所述方法例如,静脉内、皮下、肌肉内、眶内、眼内、心室内、颅内、中央囊内、脊柱内、脑池内、腹腔内、口腔、直肠、阴道、鼻内或喷雾施用。水溶液优选生理可接受的,这样除了给哺乳动物输送所需的复合物之外,溶液不会影响机体的电解质和/或容积的平衡。这样,复合物的水介质可包括通常的生理盐水(0.9%NaCl,0.15M),pH7-7.4或它的其他药用盐。
优选地,接受者应每隔两周接种三次疫苗。如果有必要,可通过随后施用这种复合物在较晚的时间内增强免疫应答。预计,通过本领域熟知的技术可经验性地为每一种应激蛋白-肽复合物制定出最合适的剂量和接种方案。
不同的细胞因子、抗生素和其它的生物活性剂也可和这种应激蛋白-肽复合物一起施用。例如,各种已知的细胞因子,即,白细胞介素-1α(IL-α)、白细胞介素-1β(IL-1β)、白细胞介素-2(IL-2)、白细胞介素-3(IL-3)、白细胞介素-4(IL-4)、白细胞介素-5(IL-5)、白细胞介素-6(IL-6)、白细胞介素-7(IL-7)、白细胞介素-8(IL-8)、白细胞介素-9(IL-9)、白细胞介素-10(IL-10)、白细胞介素-11(IL-11)、白细胞介素-12(IL-12)、干扰素α(IFN-α)、干扰素β(IFN-β)、干扰素γ(IFN-γ),肿瘤坏死因子α(TNFα)、肿瘤坏死因子β(TNFβ)、粒细胞集落刺激因子(G-CSF),粒细胞/巨噬细胞集落刺激因子(GM-CSF)和转化生长因子β(TGF-β)也可和复合物一起施用,以使生理应答最大化。然而,可以预料,其他的但尚未被发现的细胞因子对本发明也可能是有效的。此外,常规的抗生素也可和这种应激蛋白-肽复合物一起施用。但是,对合适的抗生素的选择尚依赖于所要治疗的疾病。
实施例1
在本实施例中,小鼠C57BL/6和C3H的体重大约100克,购自Jackson实验室,Bar Harbor,Me.。在小鼠皮下注射恶性肿瘤细胞,以诱发实验性肿瘤。具体地说,将恶性纺锤形细胞癌6139细胞注射到C3H小鼠的皮下,将小鼠的恶性Lewis肺癌细胞注射到C57BL/6小鼠的皮下,将小鼠的恶性B16黑色素瘤细胞注射到C57BL/6小鼠的皮下。
当肿瘤长到既能看到又能摸到大小的时候,切下肿瘤组织作样品。作为对照,也从荷瘤小鼠切下非恶性的正常组织。
然后,用以上描述的方法从切除的正常组织和肿瘤组织分离gp96-肽、Hsp90-肽和Hsp70-肽复合物。一旦分离,则把这种复合物和PBS一起回输给分离出复合物的小鼠中。通常每次实验检测6只小鼠。实验按下述方案进行:实验 回输给小鼠的组合物1 gp96-肽2 Hsp70-肽3 Hsp90-肽4 gp96-肽和Hsp70-肽5 gp96-肽和Hsp90-肽6 Hsp70-肽和Hsp90-肽7 Hsp70-肽、Hsp90-肽和gp96-肽8 只用缓冲液
在一组实验中复合物从肿瘤细胞分离,而在另一组实验中复合物从正常细胞分离。用预选的复合物20mg(全重)每隔一周给小鼠接种3次。治疗过程中,每天测量每个肿瘤的大小。4周以后处死小鼠,并对肿瘤的生长情况进行组织学检测。此外,还检测处死的小鼠有无转移。
预期的结果是,用从正常组织得到的复合物治疗的小鼠的肿瘤继续生长并且转移。相反,用从肿瘤组织得到的复合物治疗的小鼠肿瘤比对照动物的肿瘤生长慢,在某些情况下,治疗过程中的瘤块可能变小,肿瘤消退。其它的实施方案
本发明也可在不违背其精神或本质特征的情况下以其它特殊方式实施。因此,本实施方案从所有方面考虑是一种实例性的而不是限制性的,本发明的范围由所附的权利要求而不是上述说明书指出,因此权利要求的等价的含义和范围内的全部改变也将包括在内。
Claims (18)
1.一种抑制哺乳动物体内肿瘤增殖的方法,该方法包括:
给荷瘤哺乳动物施用一种组合物,该组合物包含:
(a)从肿瘤细胞分离出的免疫原性应激蛋白-肽复合物,该复合物可以有效地在哺乳动物体内起始对肿瘤的免疫应答,和
(b)药用载体,
所用的量足以在哺乳动物体内引发对肿瘤的免疫应答,由此抑制肿瘤的增殖。
2.权利要求1的方法,其中复合物中的应激蛋白是Hsp70、Hsp90或gp96。
3.权利要求1的方法,其中复合物中的肽与应激蛋白非共价结合。
4.权利要求1的方法,其中施用复合物起始T细胞介导的免疫应答。
5.权利要求4的方法,其中施用复合物起始细胞毒T细胞介导的免疫应答。
6.权利要求1的方法,其中施用给哺乳动物的复合物的剂量范围是大约1-1000微克复合物/公斤哺乳动物体重/次施用。
7.权利要求6的方法,其中所说的剂量范围是大约100-250微克复合物/公斤哺乳动物体重/次施用。
8.权利要求1的方法,其中复合物被重复地施用给哺乳动物。
9.权利要求1的方法,其中组合物与细胞因子一起施用给哺乳动物。
10.一种抑制哺乳动物肿瘤增殖的方法,该方法包括以下步骤:
(a)提供从哺乳动物切割下来的肿瘤细胞;
(b)从细胞中分离一种免疫原性应激蛋白-肽复合物,它可以有效地在哺乳动物中起始对肿瘤细胞的免疫应答;
(c)给哺乳动物施用分离的应激蛋白-肽复合物,其用量足以在哺乳动物中引发对肿瘤细胞的免疫应答,由此抑制哺乳动物中残存的任何肿瘤细胞的增殖。
11.权利要求10的方法,其中复合物中的应激蛋白是Hsp70、Hsp90或gp96。
12.权利要求10的方法,其中复合物中的肽与应激蛋白非共价结合。
13.权利要求10的方法,其中施用复合物起始T细胞介导的免疫应答。
14.权利要求13的方法,其中施用复合物起始细胞毒T细胞介导的免疫应答。
15.权利要求10的方法,其中施用给哺乳动物的复合物的剂量范围是大约1-1000微克复合物/公斤哺乳动物体重/次施用。
16.权利要求15的方法,其中所说的剂量范围是大约100-250微克复合物/公斤哺乳动物体重/次施用。
17.权利要求10的方法,其中复合物被重复施用给哺乳动物。
18.权利要求10的方法,其中复合物与细胞因子一起施用给哺乳动物。
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- 1995-04-06 AT AT95916256T patent/ATE320447T1/de active
- 1995-04-06 KR KR1019970702097A patent/KR970706013A/ko not_active Application Discontinuation
- 1995-04-06 PT PT95916256T patent/PT784477E/pt unknown
- 1995-04-06 CA CA002201498A patent/CA2201498A1/en not_active Abandoned
- 1995-04-06 EP EP95916256A patent/EP0784477B1/en not_active Expired - Lifetime
- 1995-04-06 AU AU22819/95A patent/AU709643B2/en not_active Expired
- 1995-04-06 DE DE69534874T patent/DE69534874T2/de not_active Expired - Lifetime
- 1995-04-06 EP EP06005126A patent/EP1702624A1/en not_active Withdrawn
- 1995-04-06 CN CN95196515A patent/CN1167440A/zh active Pending
- 1995-04-06 WO PCT/US1995/004347 patent/WO1996010411A1/en active IP Right Grant
- 1995-04-06 ES ES95916256T patent/ES2260762T3/es not_active Expired - Lifetime
- 1995-04-06 DK DK95916256T patent/DK0784477T3/da active
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1998
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2001
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2003
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2004
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2005
- 2005-01-20 US US11/041,099 patent/US20050163793A1/en not_active Abandoned
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Cited By (3)
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CN101297966B (zh) * | 2008-06-16 | 2012-03-21 | 黄常新 | 肠癌富伴侣分子-抗原肽复合物瘤苗及其制备方法 |
CN106279393A (zh) * | 2016-08-29 | 2017-01-04 | 拜西欧斯(北京)生物技术有限公司 | 一种gp96蛋白纯化方法 |
CN106963940A (zh) * | 2017-04-07 | 2017-07-21 | 江苏食品药品职业技术学院 | 一种增强抗肿瘤免疫应答的复合物 |
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US20020061316A1 (en) | 2002-05-23 |
JPH10506628A (ja) | 1998-06-30 |
ES2260762T3 (es) | 2006-11-01 |
US6468540B1 (en) | 2002-10-22 |
KR970706013A (ko) | 1997-11-03 |
AU709643B2 (en) | 1999-09-02 |
EP0784477A4 (en) | 2000-06-14 |
JP2008069167A (ja) | 2008-03-27 |
US20030165519A1 (en) | 2003-09-04 |
WO1996010411A1 (en) | 1996-04-11 |
DE69534874D1 (de) | 2006-05-11 |
CA2201498A1 (en) | 1996-04-11 |
US20050163793A1 (en) | 2005-07-28 |
ATE320447T1 (de) | 2006-04-15 |
JP2005015489A (ja) | 2005-01-20 |
AU2281995A (en) | 1996-04-26 |
EP0784477B1 (en) | 2006-03-15 |
EP1702624A1 (en) | 2006-09-20 |
PT784477E (pt) | 2006-08-31 |
US5750119A (en) | 1998-05-12 |
DK0784477T3 (da) | 2006-07-17 |
US6017544A (en) | 2000-01-25 |
EP0784477A1 (en) | 1997-07-23 |
DE69534874T2 (de) | 2006-12-14 |
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