CN102355908A - 纳米粒子制备的糖脂免疫抗原 - Google Patents
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Abstract
本发明是有关于一种纳米粒子制备的糖脂免疫抗原,其包含两种组分:1)大小为10-2000纳米的纳米粒子,该纳米粒子由可生物降解的多聚体组成,例如聚(乳酸)、聚(乙醇酸)、聚己酸内脂或其它聚酯类多聚体;多正脂类多聚体、聚酐类多聚体和聚磷腈类多聚体,以及由两种或两种以上的上述多聚体组成的任何共聚物。2)6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺。本发明应用的关键技术是,通过酰胺键将组分2化学结合于组分1的表面(参见图3)。本发明的目的是,为糖脂抗原提供一种新型表面结合制剂,其能够在癌症和慢性病毒感染状况的长期治疗中重复刺激免疫系统。本发明人发现,结合到纳米粒子表面的糖脂能够重复刺激NKT细胞产生IFN-γ,该IFN-γ是具有强效抗癌和抗病毒活性的细胞因子。在小鼠肿瘤转移模型中,已经发现这种新型药物具有抗癌作用。
Description
技术领域
本发明涉及一种新型纳米粒子制备的糖脂抗原,该糖脂抗原对触发免疫系统释放抗癌和抗病毒细胞因子具有较强的活性。与早先发明的糖脂抗原相比,这种新型的纳米粒子制剂不会造成免疫系统的无反应性(刺激后无应答),也可以重复用来治疗长期疾病,例如癌症和慢性病毒性疾病。
背景技术
α-半乳糖苷神经酰胺(KRN7000)(A1pha-galactosylceramide):一种有效的免疫疗剂。α-半乳糖苷神经酰胺(又称为KRN7000)是一种海绵派生的鞘糖脂(参见美国专利US 5780441)。它是NKT细胞的超拮抗剂抗原,通过Th1细胞因子(干扰素)介导机制,限制小鼠模型黑色素瘤转移并抑制乙肝病毒的复制。在一些包括癌症以及丙肝患者的临床试验中,已经验证了α-半乳糖苷神经酰胺的安全性,并且它可以强烈诱导Th1细胞因子应答。
自然杀伤T(NKT)细胞是亚群独特的淋巴细胞,其具有T细胞和NK细胞的标记和功能。NKT细胞在抗原(α-半乳糖苷神经酰胺)刺激的两小时内激活,并产生大量的Th1和Th2细胞因子。除了分泌细胞因子,NKT细胞还在启动几种类型的免疫细胞之间的细胞与细胞接触及通讯中发挥极其重要的作用,包括树突细胞、巨噬细胞、CD4T细胞和CD8T细胞。与识别肽抗原的常规T细胞相比,NKT细胞识别由非多态MHC样分子CD1d呈递的脂类抗原。
可溶性α-半乳糖苷神经酰胺诱导NKT细胞的无反应性。α-半乳糖苷神经酰胺的一个主要问题是,在一剂治疗后,它会导致NKT无应答(无反应性),这是由于α-半乳糖苷神经酰胺可以被外周血液中表达CD1d的B细胞呈递(图1A),并且在没有合适的共刺激分子的情况下刺激NKT细胞。
为了解决可溶性α-半乳糖苷神经酰胺诱导的无反应性,Dhodapkar和Steinmann研究出一种细胞治疗方法(图1B),通过静脉内注射用α-半乳糖苷神经酰胺脉冲的,由患者的外周血单核细胞离体产生的树突细胞。这种细胞治疗方法避免了NKT无反应机制,并且对诱发癌症患者的肿瘤特异性抗原CD8应答具有很强的功效。但是,细胞疗法花费高昂,并且由于病毒感染患者的组织并不符合GMP程序,所以对他们来说细胞疗法非常不切实际。因此,需要新的方法来在体内重复且有效地刺激NKT细胞。
纳米粒子制备的α-半乳糖苷神经酰胺克服了NKT的无反应性。基于上述进展,本发明人假设包装在纳米粒子中的α-半乳糖苷神经酰胺优先被树突细胞摄取,然后释放到溶酶体中(图1C)。α-半乳糖苷神经酰胺加载到溶酶体的抗原提呈分子CD1d(非MHC抗原提呈分子)上,然后再循环到细胞表面,从而激活NKT细胞。本发明的技术通过化学合成,将α-半乳糖苷神经酰胺结合到多聚体基纳米粒子表面。
利用α-半乳糖苷神经酰胺的表面结合而不是封装方法的基本原理。可以考虑两种将α-半乳糖苷神经酰胺加载到纳米粒子的方法:1)表面覆盖,也就是首先合成纳米粒子,然后将α-半乳糖苷神经酰胺结合到纳米粒子的表面;2)直接封装方法,也就是将α-半乳糖苷神经酰胺和多聚体混合到一起,并形成纳米粒子,同时α-半乳糖苷神经酰胺均匀分布在该纳米粒子内。由于NKT细胞的生物学行为,即对应α-半乳糖苷神经酰胺药品的细胞类型,本发明人决定采用前一种方法。
如图2所示,NKT细胞的激活包括增殖、收缩和记忆阶段。在增殖阶段中,NKT细胞产生抗肿瘤和抗病毒的细胞因子。在收缩和记忆阶段中,大多数NTK细胞死亡,而只有5-10%的“记忆”NKT细胞开始“休眠”,并且重新规划自身用于下次刺激。因此,α-半乳糖苷神经酰胺的连续释放不会有效地激活NKT细胞,结果表明,α-半乳糖苷神经酰胺的连续释放反而会造成NKT细胞的免疫无应答(无反应性)。根据上述机制,本发明人设计了表面覆盖的方法,所述方法使α-半乳糖苷神经酰胺在被树突细胞和巨噬细胞吞噬后迅速释放到血清中,而不是使α-半乳糖苷神经酰胺稳定地释放。相比之下,众所周知封装方法适合长期连续的释放封装的药物。封装的纳米粒子静脉内注射后聚集在肝脏或脾脏,封装药物的稳定释放可以持续长达10天的时间。因此,封装方法并不适于释放NKT配体目的。
选择生物降解多聚体基纳米粒子的基本原理。如PLGA的多聚体具有良好的生物适合性、生物降解性及商业实用性,并且早先申请中的多聚体还可以送递药物,例如蛋白、肽疫苗和疏水性抗癌药物。
可以利用暴露在纳米粒子表面的多聚体的酰基与胺基结合。然而,α-半乳糖苷神经酰胺并不包含胺基。由于已知半乳糖的6-OH基团对于α-半乳糖苷神经酰胺的抗原活性并非必需,因此,本发明人发明了糖脂类似物即6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺,其在半乳糖的6-OH基团中含有胺基。这允许形成酰胺键(参见图3)。为了避免针对任何以外的蛋白抗体应答(例如链霉抗生素蛋白-生物素系统),本发明人会直接将α-半乳糖苷神经酰胺结合到纳米聚合材料。α-半乳糖苷神经酰胺和多聚体在小鼠和人体中都不具备抗原性,并且多聚体和α-半乳糖苷神经酰胺的化学结合不会产生免疫反应抗原决定簇。
发明内容
本发明涉及一种纳米粒子制备的糖脂免疫抗原,其包含两种组分:
1)大小为10-2000纳米的纳米粒子,该纳米粒子由可生物降解的多聚体组成,例如聚(乳酸)、聚(乙醇酸)、聚己酸内脂或其它聚酯类多聚体;多正脂类多聚体、聚酐类多聚体和聚磷腈类多聚体,以及由两种或两种以上的上述多聚体组成的任何共聚物。
2)6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺。
本发明应用的关键技术是,通过酰胺键将组分2化学结合于组分1的表面(参见图3)。
本发明的目的是,为糖脂抗原提供一种新型表面结合制剂,其能够在癌症和慢性病毒感染状况的长期治疗中重复刺激免疫系统。
本发明人发现,结合到纳米粒子表面的糖脂能够重复刺激NKT细胞产生IFN-γ,该IFN-γ是具有强效抗癌和抗病毒活性的细胞因子。在小鼠肿瘤转移模型中,已经发现这种新型药物具有抗癌作用。
实施本发明的最佳模式
纳米粒子的制备,以聚(乳酸乙醇酸)(简称为PLGA)作为实例。在存在稳定的共聚物的情况下,利用纳米沉淀法制备纳米球。将来自AbsorbablePolymers International(Pelham,AL,USA)的PLGA溶于丙酮(0.5%重/容)。将该多聚体溶液逐滴添加到一种含有泊洛沙胺904(美国新泽西州帕瑟伯尼市BASF怀恩多特公司)的水溶液中。在室温下搅拌该混合物,直至有机溶剂完全干燥。将由此产生的纳米粒子分散体通过一个1μm的过滤器,并通过离心纯化。将纳米粒子重新分散到水中,在液氮中速冻并进行冻干。在-20℃的条件下储存干粉制剂。该方法提供的纳米粒子的直径为约50nm-2000nm,颗粒大小取决于PLGA的分子量、搅拌速度以及表面活性剂的浓度。
制备包含可以直接结合到PLGA纳米粒子的胺基的6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺(参见图4和图5)。
6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺与PLGA纳米粒子的结合。以1%和10%(w/w比),将6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺结合到PLGA颗粒。
试验实施例1:本发明药物的免疫刺激活性
通过纳米粒子制备的αGalCer(半乳糖基神经酰胺)在体内刺激NKT细胞:
将包含1μg的α-半乳糖苷神经酰胺的纳米粒子注射给C57BL6型小鼠,利用非结合的纳米粒子作为阴性对照。同时,本发明人研究了可溶形式的α-半乳糖苷神经酰胺。
体内重复刺激NKT细胞:从(缅因州巴港市(Bar Arbor,ME))杰克逊实验室(Jackson Laboratory)购入C57/BL6型小鼠,遵循机构方针(institutional guideline),在标准的无病原条件下将其安置在安德森癌症中心(M.D.Anderson Cancer Center)的动物设备中。将六周大的C57BL/6型小鼠用于所有的实验。每个实验利用一组三只小鼠。给每只小鼠静脉内注射(通过尾静脉)200μL含有1μgα-半乳糖苷神经酰胺或纳米粒子制备的α-半乳糖苷神经酰胺的PBS。每隔十天对小鼠进行治疗,达三次。用200ml PBS/1%二甲基亚砜作为对照。
每次刺激后,IFN-γ分泌的测量:每次药物治疗的24小时后对小鼠采血。使用BD Biosciences(San Jose,CA)的试剂盒,通过ELISA,测量血清IFN-γ。
结果:纳米粒子克服了NKT细胞的无反应性:如通过血清IFN-γ的浓度所测量的,与α-半乳糖苷神经酰胺相同,纳米粒子制备的α-半乳糖苷神经酰胺的首次注射诱发细胞因子释放,在注射24-48小时后达到峰值。如表1所示,纳米粒子的制备α-半乳糖苷神经酰胺在每次刺激后都会诱发IFN-γ分泌。与之相反,可溶性α-半乳糖苷神经酰胺仅在首次治疗时引起IFN-γ分泌,并不能在后续刺激中诱发IFN-γ分泌。因此本发明人的新型的α-半乳糖苷神经酰胺制剂可以重复刺激NKT细胞并诱发IFN-γ产生,而不会导致无反应性。
表1:αGalCer纳米粒子重复激活NKT细胞产生IFN-γ(pg/ml)。
αGalCer纳米粒子 | 不含αGalCer的纳米粒子 | αGalCer | |
×1注射 | 286±398a | 48±1 | 2601±257 |
×2注射 | 252±62 | 42±5 | 65±2 |
×3注射 | 261±102 | 45±3 | 40±11 |
a数据为每组三只小鼠的平均值和标准偏差。
试验实施例2:本发明药物的抗肿瘤活性
抗转移效果利用B16F10型黑色素瘤肺转移肿瘤模型评定本发明的抗癌功能。药物治疗后48小时,将1×105个B16F10黑色素瘤细胞通过尾静脉注射。肿瘤注射后14天,处死小鼠,并计数小鼠肺中通过转移形成的肿瘤结节(主要终末点)
结果:如图6所示,本发明具有显著的抗癌效果。
附图说明
通过参考优选实施方案的下列详细描述和附图,可获得对本发明为实现上述和其他目的而采用的结构和技术手段的最佳理解,其中:
图1是本发明的第一个实施方案的三种形式α-半乳糖苷神经酰胺的透视图;
图2是本发明的α-半乳糖苷神经酰胺作用后的NKT细胞的增殖、收缩和记忆阶段的透视图,其说明为何必须将α-半乳糖苷神经酰胺结合到纳米粒子的表面,而不是按照传统方式将其封装到内部;
图3是将α-半乳糖苷神经酰胺结合到纳米粒子表面的化学键的透视图;
图4是一种新发明的结构6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺的透视图,该结构为α-半乳糖苷神经酰胺的新型的糖脂类似物,其包含用于化学结合到纳米粒子表面的胺基;
图5是生产α-半乳糖苷神经酰胺的新型的糖脂类似物6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺的化学合成路线的透视图;
图6是本发明抗癌效果实例的透视图。
具体实施方式
下面将结合一些优选的实施方案来描述本发明。为了便于理解,以相同的标号表示优选实施例中相同的元素。
参见图1
A.可溶形式的α-半乳糖苷神经酰胺主要由B细胞呈递,其在没有共刺激信号的情况下刺激NKT细胞,并在一次药物注射后导致NKT无反应性。
B.载有αGalCer的树突细胞刺激NKT细胞,并且不会引起无反应性,但是生成用于细胞疗法的树突细胞是一个繁琐且昂贵的过程。
C.纳米粒子制备的αGalCer被设计为优先被树突细胞摄取。该α-半乳糖苷神经酰胺在溶酶体中释放并再循环到细胞表面,激活NKT细胞而不会造成无反应性。
图2表示NKT细胞的刺激包括增殖、收缩和记忆阶段。因为NKT细胞连续接触α-半乳糖苷神经酰胺会导致无应答(无反应性),所以不能以连续释放的形式送递α-半乳糖苷神经酰胺。
图3表示组分2(包含胺基)通过酰胺键结合于组分1(包含酰基)的表面。
图4表示本发明人发明的新结构6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺,它与α-半乳糖苷神经酰胺(KRN7000)的结构相似,但是可以通过其酰胺基(红色)直接结合于PLGA纳米粒子。
图5表示本发明人发明的用于生成6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺的化学合成途径。
图6表示纳米粒子制备的α-半乳糖苷神经酰胺的抗癌效果。利用B16F10黑色素瘤肺转移肿瘤模型测试纳米粒子制备的α-半乳糖苷神经酰胺药物在小鼠中的防癌活性。纳米粒子药物治疗后48小时,通过尾静脉,注射1×105个B16F10黑色素瘤细胞。肿瘤注射后14天,处死小鼠,并在利用费克特溶液(Fekete’s solution)固定后,计数小鼠肺中通过转移形成的肿瘤结节。
PBS:用PBS治疗的对照小鼠。GalCer:2μg可溶形式的αGalCer。
GalCer 200nm:2μg结合到200nm大小的聚乳酸纳米粒子的αGalCer。
GalCer 2000nm:2μg结合到2000nm大小的聚乳酸纳米粒子的αGalCer。
每组包含五只小鼠(×▲■◆*)。
Claims (9)
1.一种产品,其特征在于其在任何构型中,含有所述的单独特性组合或以任何架构特性组合。
2.一种组合物,其包含结合于纳米粒子的α-半乳糖苷神经酰胺。
3.一种组合物,其包含6-氨基乙氧基-6-脱氧-α-半乳糖苷神经酰胺。
4.根据权利要求2所述的组合物,其中所述纳米粒子包括聚乳酸、聚(乙醇酸)、聚己酸内脂或其它聚酯类多聚体;多正脂类多聚体;聚酐类多聚体和聚磷腈类多聚体,以及由两种或两种以上的上述多聚体组成的任何共聚物。
5.一种制造结合的纳米粒子α-半乳糖苷神经酰胺制剂的方法,其中通过酰胺键或其它化学键将α-半乳糖苷神经酰胺或其类似物化学结合于纳米粒子的表面。
6.一种药物组合物,其包含结合的纳米粒子α-半乳糖苷神经酰胺。
7.一种防止NKT细胞无反应性的方法,其步骤包括将疗效量的由α-半乳糖苷神经酰胺配制的结合的纳米粒子给予有需要的患者。
8.一种预防或治疗癌症、慢性病毒疾病、慢性细菌感染、自身免疫病、哮喘症、过敏症、糖尿病、动脉硬化以及其他慢性疾病状况的方法,包括将疗效量的结合的纳米粒子α-半乳糖苷神经酰胺联合蛋白/肽抗原给予有需要的患者。
9.一种蛋白/肽抗原的佐剂,其包含结合的α-半乳糖苷神经酰胺纳米粒子。
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CN104321079A (zh) * | 2012-03-19 | 2015-01-28 | 马普科技促进协会 | 糖类-糖脂类缀合物疫苗 |
CN111249451A (zh) * | 2020-01-20 | 2020-06-09 | 成都医学院 | 一种糖脂类抗原注射液及其制备方法 |
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