CN100418576C - 用于皮下施用的基于神经节苷脂的疫苗组合物 - Google Patents
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Abstract
本发明涉及疫苗组合物，所述疫苗组合物结合神经节苷脂与脑膜炎奈瑟球菌(N.meningitidis)外膜蛋白复合物(OMPC)以形成用于皮下施用的尺寸非常小的蛋白脂质体(VSSP)。本发明组合物不需要使用另加的佐剂。所述组合物能使用神经节苷脂，特别是N-AcGM3/VSSP和N-GcGM3/VSSP来进行免疫学治疗，由于所述组合物对注射部位具有较小的刺激性因而其具有优势并且可更容易地使用而使病人的不适感减少。
Description
技术领域：
本发明涉及不添加任何其它佐剂用于皮下施用的疫苗组合物，所述疫苗组合物包含神经节苷脂，用于免疫学治疗自身免疫疾病、感染疾病和肿瘤。
现有技术状况
很久以来已知将神经节苷脂用于免疫学治疗自身免疫疾病和癌症的意图，例如USA-A-4965198专利描述了神经节苷脂GM2在预防和治疗这类疾病中的用途。在提交的专利EP-A-661061和在专利US-A-6149921中描述了用以刺激或增加对抗存在于免疫原和免疫学佐剂中的神经节苷脂的抗体的疫苗组合物。
所描述的免疫原是：由N-AcetylGM3和N-GlycolylGM3神经节苷脂(现用(N-AcGM3)和(N-GcGM3)表示)与来自脑膜炎奈瑟球菌(Neisseria meningitidis)外膜蛋白复合物(OMP)结合构成的VSSP(尺寸非常小的蛋白脂质体)。
这类免疫原现称为N-AcGM3/VSSP和N-GcGM3/VSSP；其尺寸特别小并且甚至在电子结显微镜下也看不见，其是水溶性的并且具有增强的浮动能力。
对于描述于EP-A-661061和US-A-6149921中的疫苗组合物，需要使用佐剂，例如非常熟悉的弗氏不完全佐剂。
在提交的专利WO-A-02145746中描述了疫苗组合物，所述疫苗组合物含有(A)一种或更多种具有低免疫原性的抗原；(B)具有整合的神经节苷脂(主要为N-AcGM3/VSSP和N-GcGM3/VSSP)的VSSP；和(C)最后一种或更多种佐剂。
在Carr A.等人公布于Melanoma Research，2001，Vol.11，pp219-227的论文中描述了含有N-AcGM3神经节苷脂的疫苗在带有黑素瘤B16的小鼠中的抗肿瘤活性。
在该论文中也研究了免疫学佐剂特别是弗氏完全佐剂或弗氏不完全类佐剂如佐剂Montanide ISA 51存在的影响。所述疫苗通过肌内施用并且所得的结论是用N-AcGM3/VSSP和弗氏或Mont anide ISA 51中任何一种佐剂免疫的小鼠都在第八周显示IgM和IgG抗N-AcGM3的应答(表1)。相反地，不含任何佐剂的N-AcGM3/VSSP不显示任何免疫原性的应答(223页，右栏)。
因此本领域教导含有缀合有神经节苷脂的VSSP的疫苗应该与佐剂主要是弗氏(完全或不完全)或Montanide ISA 51一起配制。
然而，众所周知当其通过肠胃外施用时，这类佐剂，特别是弗氏完全佐剂，会引起某些不适的副作用例如在注射位置的慢性炎症，最终导致组织肉芽肿和无菌脓肿或溃烂性坏死。Montanide ISA 51没有那样强烈刺激(aggressive)但也能引起某些炎症。
从其在免疫学治疗自身免疫、感染和肿瘤疾病的应用上看，拥有在注射部位较少刺激和可更容易更方便地应用于病人的新的神经节苷脂疫苗组合物是非常想要的。
本发明者已发现基于神经节苷脂的与VSSP一起配制的疫苗，当其皮下施用时可不加任何佐剂使用而同时仍然存在相关的免疫学性质。
本发明的目的是包含于VSSP中的新的疫苗组合物，优选地N--AcGM3/VSSP和N-GcGM3/VSSP，所述疫苗组合物不含有任何免疫学佐剂并且通过皮下施用。
用于治疗需要增强其免疫学应答的病人的方法也是本发明的目的，所述方法包括皮下施用神经节苷脂疫苗组合物，所述疫苗组合物优选地为N-AcGM3/VSSP和N-GcGM3/VSSP，并且不含任何免疫学佐剂。
不含其它不同于神经节苷脂的抗原性组份或任何其它免疫学佐剂的N-AcGM3/VSSP和/或N-GcGM3/VSSP疫苗组合物也是本发明的目的，并且所述疫苗组合物通过皮下施用。
发明详述
本发明疫苗组合物的目的在于将一种或更多种神经节苷脂溶解或在水中分散并且将其纳入脑膜炎奈瑟球菌的OMP中(VSSP)，所述组合物能在不加任何另外的免疫学佐剂的情况下通过皮下施用刺激免疫学应答。
VSSP是高度稳定的神经节苷脂与脑膜炎奈瑟球菌外膜蛋白复合体形成(不必是共价连接)的疏水连接体。在专利EP-A-661061和US-A-6149921以及不同出版物例如，Estévez等人的Vaccine，1999；Vo l.18(1-2)：pp 190-197中详细地描述了这类神经节苷脂-蛋白质系统。这些文献也描述了获得它们的方法。
在包含于VSSP的神经节苷脂中，本发明优选的是N-AcGM3和N-GcGM3，特别优选的是N-AcGM3。
可在上述文献和综述论文：Bitton R.E等人的OncologyReports(2002)，Vol 9，267-276，和最近的通讯和科学会议例如于2002年12月9日在Havana举行的第6届拉丁美洲免疫学会议、Sáurez G等人的通讯“Phase 1 clinical trial of the gangliosidecancer vaccine N-AcetilGM3/VSSP/Montanide ISA 51 in advancedbreast cancer patients”中找到对两种缀合神经节苷脂的免疫原性以及其作为抗癌药物和获得性免疫力的刺激物的应用的详细描述。
除了神经节苷脂不含其它抗原性组份的疫苗组合物是本发明优选的目的，特别优选的是那些含有单一性免疫原性组份N-AcGM3(N--AcGM3/VSSP)和/或N-GcGM3(N-GcGM3/VSSP)的组合物，更优选是那些只含有N-AcGM3/VSSP的组合物。
本发明的疫苗组合物是最终可含有其它无毒性、非刺激性水相容性溶剂的VSSP溶液或水性分散体，所述溶剂通常在药物学上用于肠胃外使用，例如可以是聚乙二醇。
在所述溶液或水性分散体中缀合神经节苷脂的浓度不是至关重要的并且其变化范围可以是0.03％-3％(w/v)，优选地在0.04％和2.5％(w/v)之间。用于本发明涉及的疫苗皮下施用的剂量范围是50μg和2.4mg之间，优选地在200μg和2mg之间。
所述疫苗组合物的基本特征(本发明的目的)在于其设计出来在不加任何额外免疫学佐剂的情况下可进行皮下施用。
免疫学佐剂经常用于疫苗制剂中。这类佐剂以不同方式促进免疫原性作用：
-在注射部分产生抗原沉积以系统性形式放出或释放抗原。
-通过形成易于被巨噬细胞捕获的油性微滴帮助抗原到达脾和淋巴结
-直接或间接地激活参与免疫应答的细胞
-大部分已知的免疫学佐剂是：完全和不完全的弗氏佐剂、Montanide ISA、Ribi佐剂、Hunter’s TiterMax、铝盐、Gerbu佐剂、QS-21等。
令人惊奇和意外的是本发明者已发现当神经节苷脂/VSSP疫苗通过皮下施用时可完全消除佐剂。而在本领域现有技术中，对于肌内施用该现象几乎被完全排除。
因此当考虑到佐剂使用引起的局部的炎症问题时，本发明在几乎所有方面展示了无可置疑的优势。这就是使用神经节苷脂接种疫苗的简单方法，所述接种疫苗的方法对病人具有功效并且较少有刺激。
在下面的实施例中包括了比较实验细节以说明在不含免疫学佐剂的情况下所述疫苗组合物的免疫学功效。
实施例1：
通过使用描述于US-A-6149921专利中实施例3的方法制备含有2.4mg/mL N-AcGM3/VSSP的水性疫苗组合物(缓冲液Tris-HCl)。向这种免疫原组合物等份中加入等体积的Montanide ISA 51佐剂(Seppic Paris，France)。同时在另一等份中加入相同体积的Tris-HCl缓冲液。
获得两种疫苗组合物：
A：含有1.2mg/mL N-AcGM3/VSSP的水性溶液。
B：含有1.2mg/mL N-AcGM3/VSSP的W/O乳液。
选择50只体重在18-20g之间的C57BL/6雌性小鼠并将其分成5个实验组，每个组10只动物。
第1组(对照)动物在第0、14、28和42天时用0.1mL磷酸缓冲盐溶液(PBS)进行肌内接种疫苗。
第2组动物在第0、14、28和42天时用0.1mL疫苗组合物B(120μg N-AcGM3/VSSP)进行肌内接种疫苗。
第3组(对照)动物在第0、14、28和42天时用0.1mL(PBS)进行皮下接种疫苗。
第4组动物在第0、14、28和42天时用0.1mL疫苗组合物B(120μg N-AcGM3/VSSP)进行皮下接种疫苗。
第5组动物在第0、14、28和42天时用0.1mL疫苗组合物A(120μg N-AcGM3/VSSP)进行皮下接种疫苗。
在第63天时对所有组中的小鼠用5×103个黑素瘤MB16F10细胞(0.2mL)皮下进行攻击。
自第0天开始对所述动物进行单个考察并且每星期两次确定下列参数：肿瘤体积、存活和进展时间。
获得的结果如下：
肿瘤体积：
图1显示来自各实验组的肿瘤生长动力学曲线。使用Mann-Whitney(two-tailed)U测试法评估成对组群的肿瘤体积值之间的统计学显著性，所述肿瘤体积值来自用肿瘤攻击后第33天的单个动物。
该统计学方法特别适合于对这类具有涉及生物学事件的数据天然离散的实验进行评估。
P值表示与从样品中计算而来的实际值相关的概率并且允许确定通过stadigraph和确证零假设(p＞0.05)的实际数据计算而来的α值的近似程度(显著性)。
结果显示于图1和表1
表1.-疫苗接种对肿瘤体积的效果
a：作为第2组与第1组对比结果的p值
b：作为第4组与第3组对比结果的p值
c：作为第5组与第3组对比结果的p值
从图1和表1中可看出，相对于实验中的其它组群和相应的对照，皮下接种本发明组合物的第5组小鼠显示肿瘤体积显著减少。
存活
该参数用以评估在受到如MB16般致命肿瘤攻击时，接种疫苗增加受免疫动物的寿命的能力。以天数测量所述参数，与非处理组动物存活天数相比较。使用Log-Rank测试法确定统计学显著性。
图2和表2显示获得的值。表2中短语参照组X表示被用来与各组进行比较的组。
表2.根据Log-Rank测试法的存活时间统计
这些结果表明在接种肿瘤后，第5组内用本发明制剂进行皮下接种疫苗的小鼠显示最长的存活时间。
进展时间
进展时间是用以估计各个体动物中的肿瘤变得明显所需时间的参数，从接种的时刻开始测量。结果显示于表3中：
表3.根据Log-Rank测试法的进展时间统计
对于在结束实验时没有产生任何肿瘤的小鼠，其进展时间被认为是60天。
延长进展时间的效果很明显是非常想要的衡量疫苗对抗癌症方面显著差异的参数，并且根据表3中的观察结果，用本发明VSSP组合物皮下接种的第5组动物表现更为相关的正结果。
肿瘤消退
在对应于使用和不使用佐剂皮下接种疫苗的第4和第5组中，观察到具有肿瘤消退的动物。第4组中的动物在接种后第19天便可测量到肿瘤并且肿瘤实际上保持无任何增长直至第35天发现减小。第5组的小鼠在接种后第26天通过触诊发现有肿瘤存在并且据报道在第29天开始变小。
总体评估
实验结果显示本发明的疫苗组合物延长了存活时间和进展时间，同时相对于使用PBS的对照组显著地降低了肿瘤生长速度。
另一方面含有Montanide ISA 51佐剂的疫苗组合物在肌内施用时只显示保护作用，而在皮下施用时完全无效。
总之相对于用含有Montanide类型佐剂的疫苗组合物通过肌内方式获得的结果，用本发明疫苗组合物进行皮下接种疫苗显示更优的结果。
附图简述
图1展示的图表表示在5组接受不同疫苗处理和恶性肿瘤攻击的动物中肿瘤体积的演变。
图2展示的图表允许评估5组实验动物的存活参数。
Claims (6)
1. 疫苗组合物，所述疫苗组合物含有一种或更多种包含于VSSP中的神经节苷脂的溶液或水性分散体，其中所述包含于VSSP中的神经节苷脂选自N-AcGM3和N-GcGM3，所述疫苗组合物不含任何免疫学佐剂即能通过皮下施用而刺激免疫应答。
2. 权利要求1的疫苗组合物，其中包含于VSSP的神经节苷脂是N-AcGM3。
3. 权利要求1的疫苗组合物，其中所述疫苗组合物不含有其它不同于神经节苷脂的抗原。
4. 权利要求2-3任一项的疫苗组合物，其中所述组合物只含有免疫学组份N-AcGM3/VSSP和N-GcGM3/VSSP。
5. 权利要求4的疫苗组合物，其中所述组合物只含有组份N--AcGM3/VSSP。
6. 权利要求1-5中任一项的疫苗组合物在制备药物组合物中的应用，其中所述药物组合物用于皮下施用，以治疗需要增强其免疫应答的患者。
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| CN1122247A (zh) * | 1993-12-29 | 1996-05-15 | 分子免疫中心 | 引发抗n-羟乙酰化神经节苷脂免疫反应的疫苗组合物及其应用 |
| US6149921A (en) * | 1993-12-29 | 2000-11-21 | Centro De Inmunologia Molecular | Vaccine compositions for eliciting an immune response against N-acetylated gangliosides and their use for cancer treatment |
| CA2399325A1 (en) * | 2001-08-21 | 2003-02-21 | National Research Council Of Canada | Carbohydrate-based whole cell cancer vaccines |
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| US6149921A (en) * | 1993-12-29 | 2000-11-21 | Centro De Inmunologia Molecular | Vaccine compositions for eliciting an immune response against N-acetylated gangliosides and their use for cancer treatment |
| CA2399325A1 (en) * | 2001-08-21 | 2003-02-21 | National Research Council Of Canada | Carbohydrate-based whole cell cancer vaccines |
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