CN112334143A - 来自巨噬细胞和经辐射白细胞共培养物的上清液,用于控制肿瘤的进展或恢复抗肿瘤免疫力 - Google Patents
来自巨噬细胞和经辐射白细胞共培养物的上清液,用于控制肿瘤的进展或恢复抗肿瘤免疫力 Download PDFInfo
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Abstract
本发明涉及通过药物制剂解决促肿瘤炎症的方法。从全血获得的血沉棕黄层中分离出白细胞,并将一部分白细胞置于接受分化因子的第一袋中,并在提供成分确定的新鲜培养基之前,在保持细胞活力的条件下保存几天,之后,将所述白细胞部分再静置几天,导致巨噬细胞的产生。将另一部分白细胞置于第二袋中并在其中进行辐射。然后将两个袋混合,并回收上清液以用作药物制剂。本发明还涉及在人类医学和兽医学中单独或与其他疗法组合用于治疗癌症的用途。
Description
技术领域
本发明涉及通过药物制剂解决促肿瘤炎症的方法。
背景技术
炎症反应是生物过程,是有机体自然防御反应的一部分,但也与多种病理反应有关。
特别地,炎症引起诸如肠的炎症疾病之类的炎症疾病,例如克罗恩病和类风湿性关节炎,但是它也与肥胖、糖尿病和例如抑郁症的神经功能障碍的发病机理有关。促炎因子,例如促炎细胞因子,参与并支持肥胖、肿瘤发生和癌症发展过程中的炎症。实际上,慢性炎症在促进免疫系统对肿瘤的治疗失败和维持肿瘤的发展中起重要作用。炎症仍然是癌症的急性危险因素。
慢性炎症可能与炎症反应的部分或不完全解决有关,而遗传、环境或感染因素可能会促进这种炎症。慢性炎症特别涉及由病毒感染引起的癌症的进展,例如与乳头瘤病毒(HPV)相关的炎症,这会增加罹患宫颈癌的风险,以及与乙型肝炎病毒和丙型肝炎病毒及肝癌的出现相关的炎症,或者还有与幽门螺杆菌以及腺癌或淋巴瘤的发展有关的炎症。自身免疫性疾病(例如克罗恩病或溃疡性结肠炎)中的慢性炎症也与患结直肠癌的风险增加相关。最后,暴露于刺激物或肥胖引起的炎症也促进了促肿瘤活动。
因此,在引起炎症的有机体的攻击过程中,触发炎症的解决很重要。如果炎症不能完全解决,则炎症可能会变得慢性,导致上述病理。
如果炎症介质是抗肿瘤监测必不可少的,特别是在癌性免疫编辑阶段,允许建立适应性的抗肿瘤免疫应答,后者可以发挥促肿瘤的作用,特别是TNF(肿瘤坏死因子)。实际上,肿瘤炎症微环境可以促进肿瘤生长、血管生成、转移的侵袭和形成。肿瘤的发展是由于癌细胞的增殖增加,它们在肿瘤微环境中对通过凋亡死亡的抗性和血管生成导致的肿瘤细胞侵袭的结果。
例如,TNF可促进肿瘤中调节性T细胞(Tregs)以及骨髓来源的抑制性细胞(MDSCs)的吸引,从而进一步减弱抗肿瘤应答。自相矛盾的是,虽然诱导肿瘤细胞免疫原性死亡的细胞毒性疗法已经可以证明免疫系统在选择常规抗癌治疗方法中的主要作用,但现在将描述相同的方式,将通过这些疗法杀死的死细胞与继发性促肿瘤作用相关联,这些死亡细胞会刺激肿瘤的生长。
在慢性炎症疾病中,解除炎症的解决也仍然是重新激活炎症的自然解决的治疗选择,即允许阻止炎症应答。目前,已显示出中和或破坏炎症因子的靶向治疗方式具有局限性。此外,仅通过中和炎症因子就能够控制复杂的病理学例如克罗恩病似乎是错觉,这仅仅是病理学的结果。但是,通过重新建立有效的解决方案来解决炎症可能使治疗炎症反应的核心成为可能。
因此,促进炎症的解决并改善由抗癌疗法杀死的肿瘤细胞的消除已成为癌症护理中的主要挑战。以相同的方式靶向于炎症的解决可以使控制炎症病理的根本原因成为可能。
发明内容
本发明特别涉及通过施用可触发炎症解决的解决组(résolutome),即促解决因子(facteurs pro-résolutifs)的组合,通过抑制肿瘤逃避免疫监测及其发展以及通过重建有效的抗肿瘤免疫力,来预防癌症的方法。
专利EP 2 941 257描述了一种用于预防或治疗病理性免疫应答的药物制剂。该药物制剂包含可从吞噬细胞与凋亡细胞共培养物获得的上清液。在该专利中,药物制剂用于治疗诸如自身免疫性疾病的病状,例如类风湿性关节炎(RA)或肠的慢性炎症疾病(慢性炎症肠病-CIBD)。这种药物制剂也被用于治疗移植物抗宿主病(GVHD)。
本发明具有不同的目的,即通过经辐射的巨噬细胞和白细胞的共培养物获得的上清液解决促肿瘤炎症的方法;与专利EP 2 941 257中描述的上清液相比,该上清液以出乎意料的方式具有许多优点。特别是,与上述专利的上清液相比,它提供了更多数量的促解决因子(图1a);它还具有生物学优势(图1b)。
根据本发明的药物制剂可以在解决失败的情况下使用,以重新建立有效的解决并允许阻止炎症反应。因此,本发明的目的特别是通过上述药物制剂解决慢性炎症和解决促进肿瘤生长的炎症。
本发明首先基于以下事实:靶向于促肿瘤炎症的解决使得可以恢复抗肿瘤免疫应答并停止慢性抗炎反应。
在此阶段应记住,当肿瘤开始形成时炎症是有益的,但是在此之后具有促进治疗失败的负面作用,这是本发明旨在控制的。
根据本发明的方法包括使用通过从白细胞产生源自单核细胞的巨噬细胞并将其与先前已经辐射过的白细胞一起储存而获得的上清液,从而使它们能够共培养,并收集细胞产生的上清液中包含的因子。
在以下描述中,旨在解决炎症的根据本发明的药物制剂的上清液将被称为“瑞宗维克斯(Resolvix)”。
这些因子的注入使得有可能在多种实验模型(关节炎、炎症性肠病-IBD、结肠炎、实验性自身免疫性脑脊髓炎)中解决活动性炎症反应,而且还可以促进组织修复。以一种有趣的方式,这种方式还使恢复抗癌免疫力成为可能,从而促进肿瘤消退。
作为非限制性实施例,现在将描述根据本发明的生产上清液(Resolvix)的方法。
通过密度梯度从全血中在D0分离白细胞,洗涤,重悬浮在本技术领域的本领域技术人员通常使用的成分确定的培养基(defined culture medium)中,该培养基具有盐、氨基酸和维生素基础,例如像称为RPMI 1640的培养基,然后将60%分配到第一袋中,将40%分配到第二袋中。然后第一袋接受分化因子,可以是M-CSF(巨噬细胞集落刺激因子),并保存在保持细胞活力的条件下,例如在37℃,5%CO2下持续几天,优选3天,然后再次接受上述成分确定的新鲜培养基。然后将所述第一袋保持原样,在保持细胞活力的条件下,例如在37℃,5%CO2的条件下保存几天,优选4天后,导致产生巨噬细胞。第二袋又被保存在低温下,优选地在低于+10℃的温度下,并且更优选地在+5℃以下。在第7天,第二袋得到辐射,例如通过X射线,然后将第一袋和第二袋去除它们的培养基。然后将第二袋放入本领域技术人员已知的具有盐、氨基酸和维生素基础的分泌培养基中,例如像被称为MEM的一种,并转移到第一袋中以在保持细胞活力的条件下存储,例如在37℃,5%CO2下。在此存储期间,共培养发生导致产生条件化的上清液,这可能需要几天,优选2天。在本说明书中,“条件化的上清液”是含有源自细胞的因子的上清液。在此共培养期之后,将上清液收集在新的袋中,过滤,然后保存在非常低的温度下,优选在-80℃的范围内。然后根据上述方法,将该上清液与产生自其他供血者产生的血沉棕黄层的上清液有利地混合,然后将该混合物分装成单位剂量,冻干或不冻干,然后储存。
附图说明
以更精确的方式,使用肿瘤生长的体内模型。获得的结果显示在以下附图中:
图1:产生名为Resolvix的上清液的新生产方法的生物学和活性比较。这种新的生产方法使得有可能获得更多数量的促消退因子,例如通过ELISA试验定量的抗炎细胞因子TGF-β(a)和通过生物学测试评价的抑制植物血凝素刺激的单核细胞中TNF产生的生物学优势(b)。来自旧方法的35个样品和89个新方法样品的数据表示为抑制TNF产生的百分比,以平均值的平均值+/-标准误表示。***=p<0.00l,****=p<0.0001,未配对的学生t检验(test t de Student non apparié)。在该图中,旧方法涉及产生前述专利EP 2 941 257中所述的上清液的方法。
图2:追踪肿瘤生长。肿瘤细胞系EL4-Luc+(注射荧光素酶后)发出的生物荧光已在患有白血病的小鼠C57B1/6中进行了定量,在注射白血病细胞后在D0或D7时用瑞宗维克斯(Resolvix)处理或未处理过(a)。由每组5只动物的两个组的代表性经验得出的数据,表示为组平均值+/-平均值的标准误。*=p<0.05,****=p<0.0001,相对于载体(véhicule),双向方差分析测试(test 2way AOVA)。显示了在注射肿瘤细胞21天后获得的5只未经治疗的小鼠(载体)和5只在D0接受Resolvix的小鼠的生物荧光的代表性图像(b)。发出的荧光强度(从深蓝色到黄色)与已增殖的肿瘤细胞数量成正比。
图3:Resolvix治疗后肿瘤的演变。肿瘤体积(以mm3计)通过随时间向小鼠C57B1/6的右腹侧注射肿瘤系B16-OVA(a)或肿瘤系EL-4(b)来评估。在注射癌细胞当天(J0)或7天后(J7)注射Resolvix。由每组5只动物的6个组的2个实验(a和b)代表得出的数据,表示为平均值+/-平均值的标准误。*=p<0.05,**=p<0.001,以及***=p<0.001,相对于载体,双向方差分析测试。
图4:Resolvix存在下癌细胞系EL-4体外细胞增殖的演变。通过3天的每天的细胞计数来评估细胞增殖。数据来自两个有代表性的实验,表示为一式三份的平均值+/-平均值的标准误。*=p<0.05,****=p<0.000l相对于Resolvix,双向方差分析测试。
图5:免疫缺陷小鼠中的实体瘤的Resolvix治疗。在不同的时间确定由癌细胞系EL-4皮下注射到免疫缺陷小鼠RAG-γ/c的右腹侧皮下的肿瘤体积(以mm3计)。在注射癌细胞之日(D0)或7天后(D7)注射Resolvix。由每组5只动物的实验得出的数据,表示为平均值+/-平均值的标准误。
具体实施方式
所述数据以有趣的方式证明了在荷有T淋巴瘤白血病(肿瘤细胞系EL4-荧光素酶+能够通过生物成像追踪肿瘤生长)的小鼠中注射Resolvix导致肿瘤生长消退(图1)。
在实体瘤(皮下注射肿瘤细胞系EL-4或B16-OVA)的第二种模型中也获得了相同的观察结果,其中注射Resolvix使得减小肿瘤的大小成为可能(图2)。
这些结果表明Resolvix治疗可以通过直接的细胞毒性作用直接影响肿瘤细胞系的体内生长。为了评估这种可能性,已经进行了其他体外实验并且已经显示在Resolvix存在下的肿瘤细胞EL-4的培养反而促进了系的生长(图3)。这些数据表明,用Resolvix治疗后观察到的体内肿瘤生长抑制与药物对肿瘤细胞的直接细胞毒性作用无关。
此外,治疗的效果并不直接作用于肿瘤细胞这一事实表明,抗肿瘤免疫力的恢复是由于治疗所致。确实,在缺乏免疫系统的情况下,即在免疫缺陷的C57B1/6RAG-γ/c肿瘤小鼠中,Resolvix治疗对体内肿瘤生长没有作用(图4)。
在移植肿瘤细胞时(D0)或移植后7天(D7),在肿瘤模型中进行Resolvix注射。因此通过在这些不同时间控制炎症,控制肿瘤的生长并重建抗肿瘤免疫力是可能的。
因此,以上表明,根据本发明的Resolvix的使用表现出使得恢复抗肿瘤免疫力和/或停止肿瘤进展成为可能。更一般地,根据本发明的药物制剂可以用于靶向、控制、抑制、解决与癌症有关的炎症,因此使得如刚刚提到的恢复抗肿瘤免疫力和/或停止肿瘤进展成为可能。
根据本发明的上清液可以特别地单独地或与其他疗法组合,在人类医学或兽医学中用于癌症的治疗。特别是以淋巴瘤、白血病、肉瘤和癌为代表的癌症,更准确地说,是T细胞淋巴瘤、B细胞淋巴瘤、黑色素瘤和结肠癌。
Claims (11)
1.一种用于控制肿瘤进展或恢复抗肿瘤免疫力的药物制剂,所述药物制剂包含从巨噬细胞和经辐射的白细胞之间的共培养物获得的上清液。
2.根据权利要求1的药物制剂,其特征在于所述巨噬细胞是白细胞。
3.根据权利要求1和2所述的药物制剂,其特征在于所述白细胞从全血产生的血沉棕黄层分离。
4.根据权利要求3所述的药物制剂,其特征在于所述白细胞的一部分得到洗涤,重悬于成分确定的培养基中,然后置于接受分化因子的第一袋中,并保存在能维持细胞活力数天,优选3天的条件下,随后再次接受新鲜的成分确定培养基,然后将其再静置几天,优选4天,导致巨噬细胞的产生。
5.根据权利要求3所述的药物制剂,其特征在于所述白细胞的另一部分置于第二袋中,所述第二袋保持在低温下,优选地低于+10℃,并且更优选地低于+5℃。
6.根据权利要求5所述的药物制剂,其特征在于所述第二袋在除去其经辐射的培养基后,所述第二袋不含其培养基,然后在保持细胞活力的条件下放入分泌培养基中。
7.根据权利要求6所述的药物制剂,其特征在于所述第二袋被转移到所述第一袋中,所述第一袋先前去除了其培养基,所述两个袋的集合被保存在保持细胞活力的条件下一段时间,用来形成白细胞之间的共培养物,导致产生条件化的上清液。
8.根据权利要求7所述的药物制剂,其特征在于将所述共培养物的所得上清液收集在新的袋中,过滤并保持在非常低的温度下。
9.根据权利要求1至8中任一项所述的药物制剂,其特征在于所述药物制剂或者单独或者与其他抗癌疗法一起在治疗癌症中的用途。
10.根据权利要求9所述的药物制剂,其特征在于所述癌症特别是淋巴瘤、白血病、肉瘤和癌的代表,以及更准确地是T细胞淋巴瘤、B细胞淋巴瘤、黑色素瘤和结肠癌。
11.根据权利要求9或10中任一项使用的药物制剂,其特征在于所述药物制剂是根据权利要求1至8中任一项从几种白细胞的源获得的上清液混合物的结果。
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