CN109069634A - 能够抑制外泌体中的遗传功能的复合物以及癌症增殖和/或转移抑制剂 - Google Patents
能够抑制外泌体中的遗传功能的复合物以及癌症增殖和/或转移抑制剂 Download PDFInfo
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Abstract
本发明提供一种缀合物,其包含靶向外泌体表面抗原的抗体或抗体片段,以及基因或其表达产物的抑制剂,其中抗体或抗体片段和基因或其表达产物的抑制剂直接或经由连接基团共价结合或非共价结合。
Description
技术领域
本发明涉及能够抑制外泌体的遗传功能的缀合物、以及癌症增殖和/或转移抑制剂。
背景技术
最近的研究发现了一种机制,其中包括T细胞、血小板、上皮细胞、免疫细胞或癌细胞的各种细胞释放具有40至100nm直径的称为外泌体的囊泡,从而将信息传递给远处细胞。
具体地,已经提出了一种新的机制,其中癌细胞增殖和转移能力由包含在血液中分泌的外泌体中的基因(例如miRNA)的表达产物支配,并且该机制已经引起了极大的关注。
为了抑制在外泌体中的miRNA的功能,通常使用采用具有与miRNA互补的序列的修饰核酸(反义核酸)的方法(非专利文献1至3)。然而,由于血液中的miRNA被包封在外泌体中,因此即使通过向血液中施用反义核酸也难以直接靶向miRNA。
使用外泌体作为药物递送系统的许多方法是已知的(非专利文献3至5,以及专利文献1和2)。
引文列表
专利文献
专利文献1:JP2010-285426A
专利文献2:JP2014-185090A
非专利文献
非专利文献1:G.Hutvagner,M.J.Simard,C.C.Mello and P.D.Zamore,PLoSBiol.,2004,2,E98。
非专利文献2:U.A.Orom,S.Kauppinen and A.H.Lund,Gene,2006,372,137-141
非专利文献3:S.Davis,B.Lollo,S,Freier and C.Esau,Nucleic Acids Res.,2006,34,2294-2304
非专利文献4:Lai CP,Mardini O,Ericsson M,Prabhakar S,Maguire CA,Chen JW,etal.Acs Nano.2014;8(1):483-94
非专利文献5:Smyth T,Petrova K,Payton NM,Persaud I,Redzic JS,Graner MW,etal.Bioconjugate Chem.2014;25(10):1777-84。
发明内容
技术问题
本发明的主要目的是提供一种抑制外泌体中所含的基因(如miRNA)或其表达产物的功能的新方法。
技术问题
本发明提供以下缀合物和/或癌症增殖和/或转移抑制剂。
项1.一种缀合物,其包含靶向外泌体表面抗原的抗体或抗体片段,以及基因或其表达产物的抑制剂,其中抗体或抗体片段和基因或其表达产物的抑制剂直接或经由连接基团共价结合或非共价结合。
项2.根据项1所述的缀合物,其中,外泌体表面抗原是CD9、CD63、CD81或CD147。
项3.根据项1或2所述的缀合物,其中抗体或抗体片段用含有选自由半胱氨酸、精氨酸、赖氨酸和鸟氨酸组成的组中的至少一种氨基酸的肽修饰,并且基因或其表达产物的抑制剂经由共价键(S-S键)、配位键或非共价键与肽结合。
项4.根据项3所述的缀合物,其中,肽还包含甘氨酸或丙氨酸。
项5.根据项3所述的缀合物,其中,肽为聚赖氨酸或聚精氨酸。
项6.根据项5所述的缀合物,其中,肽为聚精氨酸。
项7.根据项1至6中任一项所述的缀合物,其中,基因或其表达产物的抑制剂是抗miRNA核酸,并且抗miRNA核酸是通过与外泌体中含有的miRNA形成互补链来抑制miRNA功能的核酸。
项8.根据项1所述的缀合物,其中抗体或抗体片段是抗CD63抗体。
项9.根据项1所述的缀合物,其中,基因或其表达产物的抑制剂是外泌体中含有的miRNA或基因的抑制剂。
项10.根据项1所述的缀合物,其中,基因或其表达产物的抑制剂是miRNA抑制剂。
项11.根据项1所述的缀合物,其中抗体或抗体片段是单克隆抗体、单链抗体、Fab、Fab'、F(ab')2、Fv或scFv。
项12.一种癌症增殖和/或转移抑制剂,包含项1至11中任一项所述的缀合物。
本发明的有益效果
本发明能够有效地抑制特别是参与癌症转移和增殖的外泌体中所含基因的功能。
附图说明
图1:本发明的缀合物的作用机理的示意图。
图2:示出实施例1的荧光标记抗体的制备方案的示意图。
图3:示出实施例1的结果的共聚焦激光显微镜图像。
图4:示出实施例2的缀合物的制备方案的示意图。
图5:示出实施例2和比较例1的结果的共聚焦激光显微镜图像。标题为“抗CD63IgG+RNA(Cy5)”的上排示出比较例1的结果,标题为“抗CD63IgG-9r+RNA(Cy5)”的下排示出实施例1的结果。鬼笔环肽是特异性结合至构成细胞骨架的聚合肌动蛋白(F-肌动蛋白)的寡肽,并用于细胞骨架的染色。
图6:示出实施例3的制备方案的示意图。
图7:将抗CD63抗体/抗miR缀合物的微小RNA功能抑制作用掺入细胞中。
图8:外泌体miR21依赖的细胞增殖抑制。
图9:抗CD63抗体/抗miR核酸缀合物在体内的作用。
具体实施方式
基因或其表达产物的抑制剂是外泌体中所含基因的功能的抑制剂。其实例包括低分子化合物、miRNA抑制剂、DNA抑制剂、mRNA抑制剂、tRNA抑制剂、rRNA抑制剂、piRNA抑制剂、非编码RNA抑制剂、适体、抗体、F(ab')2片段、单链抗体片段、Fv片段、单链Fv片段、亲合体、纳米抗体和选择性抗体支架(例如双抗体)。
在本发明的一个优选实施方式中,基因或其表达产物的抑制剂的实例包括低分子化合物和抗miRNA核酸。低分子化合物的实例包括顺铂、5FU(5-氟尿嘧啶)、多柔比星、放线菌素、丝裂霉素、环磷酰胺、美法仑等。
抗体或抗体片段的实例包括单克隆抗体、单链抗体、Fab、Fab'、F(ab')2、Fv或scFv。
miRNA(微小RNA)是包含在外泌体中的RNA,具有约15至30个碱基,特别是约18至25个碱基。在本发明中,“外泌体”广泛地包括从哺乳动物细胞释放的囊泡。动物的实例包括人、猴、牛、绵羊、山羊、马、猪、兔、狗、猫、大鼠、小鼠、豚鼠等,特别优选人。哺乳动物细胞的实例尤其包括肿瘤细胞、树突细胞、巨噬细胞、T细胞、B细胞、血小板、网织红细胞、上皮细胞、成纤维细胞等,特别是肿瘤细胞。外泌体的直径为约30至200nm,优选约30至100nm。
作为抗体或抗体片段的靶标的外泌体表面抗原的实例包括CD9、CD63、CD81和CD147。作为抗体或抗体片段的靶标的优选的外泌体表面抗原的实例包括CD9和CD63,更优选CD63。
本发明的缀合物包含作为必需成分的靶向外泌体表面抗原的抗体或抗体片段,以及基因或其表达产物的抑制剂。
靶向外泌体表面抗原的抗体或抗体片段的实例包括抗CD9抗体、抗CD63抗体、抗CD81抗体、抗CD147抗体和这些抗体的片段。优选的实例包括抗CD9抗体、抗CD63抗体及其抗体片段。进一步优选的实例包括抗CD63抗体及其抗体片段。
通过具有与外泌体中包含的miRNA互补的序列,抗miRNA核酸与miRNA形成互补链,从而抑制miRNA的功能。抗miRNA核酸可以仅由与miRNA互补的序列组成,或者可以在与miRNA互补的序列的5'末端或3'末端添加任何序列。加入的序列中的碱基数为50或更少,优选40或更少,更优选20或更少,进一步优选10或更少,特别优选5或更少。最优选的抗miRNA核酸仅由与靶标miRNA互补的链组成。抗miRNA核酸是DNA、RNA或核酸衍生物,优选为RNA。核酸衍生物表示这样的衍生物,其中例如碱基部分、核糖部分、磷酸二酯键部分的原子(例如,氢原子,氧原子)或核酸的官能团(例如,羟基、氨基)被另外的原子(例如氢原子、硫原子)、官能团(例如,氨基)或C1-6烷基取代,或者被保护基团(例如甲基或酰基)保护,或其中这些部分被非天然组分(例如肽键)取代的那些。此类核酸衍生物的实例包括碱基部分通过肽键连接的肽核酸(PNA)、甘油核酸(GNA)、苏糖核酸(TNA)、桥接核酸(BNA)、碱基的氨基的氢原子被C1-6烷基取代的核酸、核糖部分的羟基的立体构型被修饰的核酸、具有磷酸二酯键部分中的氧原子被硫原子取代的硫代磷酸的核酸。
抗miRNA核酸可以含有一个与目标miRNA互补的序列,或者两个或多个(例如,2-10个,优选2、3、4或5个)直接或通过适当碱基与目标miRNA互补的序列。此外,作为互补序列,可以包括多个一种类型的序列,或多个不同种类的互补序列。抗miRNA核酸可以是单链核酸、或双链核酸。双链核酸的实例包括dsRNA和siRNA。具有发夹结构的RNA(例如shRNA)也包括在抗miRNA核酸中。双链核酸包括DNA-RNA杂合体。
待在功能上被抑制的外泌体的基因或其表达产物是特别是参与癌症增殖和转移的基因。
基因或其表达产物的抑制剂(例如miRNA抑制剂)、抗体或抗体片段可以直接或通过连接基团共价结合,或者可以非共价结合。非共价键的实例包括离子键、配位键、疏水相互作用等。例如,当采用具有至少一个半胱氨酸残基的肽修饰抗体或抗体片段时,具有SH基团的基因或表达产物的抑制剂可以经由半胱氨酸的SH基团通过S-S键共价结合,或者可以由金属离子通过-S-(金属离子)-S-配位键结合。半胱氨酸的数量可以是一个、或两个或更多个。当采用具有至少一个精氨酸残基的肽修饰抗体或抗体片段时,阴离子基因的抑制剂(例如抗miRNA核酸或其表达产物)可以通过离子键非共价结合到精氨酸残基的阳离子。此外,如果采用具有赖氨酸残基或鸟氨酸残基的肽修饰抗体或抗体片段,则阴离子基因(例如抗miRNA核酸或其表达产物)可以通过离子键非共价结合到赖氨酸残基或鸟氨酸残基的阳离子,或者也可以通过赖氨酸残基或鸟氨酸残基的末端氨基(NH2)直接或通过适当的连接基团共价结合。
直接或通过连接基团与抗体或抗体片段结合的肽优选是由选自赖氨酸(Lys,K)、精氨酸(Arg,R)和鸟氨酸(Orn)的碱性氨基酸构成的肽。肽可包括用于调节连接基团长度的甘氨酸残基或丙氨酸残基。碱性氨基酸更优选为赖氨酸(Lys,K)或精氨酸(Arg,R)。优选实施方式中的肽是聚精氨酸或聚赖氨酸。肽的氨基酸数量没有特别限制,只要阴离子基因或其表达产物的抑制剂(例如miRNA抑制剂)的键是可能的即可;该数量为例如4至50,优选5至40,更优选6至30,进一步优选7至25,特别优选8至20。例如,当肽仅由碱性氨基酸构成时,与肽结合的阴离子基因或表达产物的抑制剂(例如,miRNA抑制剂)的数量为1或2;优选地,肽和阴离子基因或其表达产物的抑制剂以1:1结合。与单一抗体或抗体片段结合的肽的数量是1至10、1至8、1至6、1至4、或1至2。通过将多个肽结合到抗体,可以获得包含多种miRNA抑制剂的缀合物。据说外泌体中的miRNA超过200种,当存在大量的目标miRNA时,可以将多个肽结合到单一抗体上,从而结合多种miRNA抑制剂。此外,当外泌体中存在大量的表面抗原(如CD9、CD63、CD81或CD147)时,可增加抗体的数量。
在本说明书中,“用肽修饰的抗体或抗体片段”是指肽与抗体或抗体片段共价结合。肽结合的位点的实例包括抗体或抗体片段的恒定区(CH1,CH2,CH3)以及诸如Fc区的恒定区。肽与抗体或抗体片段的结合可以根据标准方法进行,例如,根据下面所示的方案1进行。
方案1
其中Ab是抗体或抗体片段;与Ab结合的NH2是在不显著影响Ab键的区域(例如,恒定区,如CH1、CH2、CH3或Fc区)中的氨基酸的氨基;P1是含有选自由半胱氨酸、精氨酸、赖氨酸和鸟氨酸组成的组中的至少一个氨基酸的肽,或基因或其表达产物的抑制剂。
使抗体或抗体片段(1)与化合物(2)反应,由此得到脒化合物(3),使脒化合物(3)与化合物(4)反应,由此得到抗体或抗体片段(5),在抗体或抗体片段(5)中,基因或其表达产物的抑制剂或肽被结合。获得基因或其表达产物的抑制剂或肽结合的抗体或抗体片段(5)的反应条件可由本领域技术人员通过参考ACS Chem.Biol.(2011年,6月,962-970页)等的公开内容而容易地确定。以上所示的方案1仅是一个实例,基因或其表达产物的抑制剂或肽直接或通过连接基团共价结合到抗体或抗体片段的任何状态都包括在本发明中。
用于基因或其表达产物的抑制剂、肽、和抗体或抗体片段的结合的连接基团的实例包括-O-、-CO-、-CONH-、-NHCO-、-NH2-、-(OCH2CH2)n-,在末端具有马来酰亚胺基和琥珀酰亚胺基的二价连接基团等。
将肽结合的抗体或抗体片段与抗miRNA核酸在水或类似溶剂中混合,从而形成缀合物。
如图1所示,当静脉内注射本发明的缀合物时,缀合物的抗体或抗体片段位点结合到外泌体。此时,基因或其表达产物的抑制剂(图1中的抗miR)存在于外泌体外。然而,当将外泌体掺入细胞中时,抑制剂也与外泌体一起掺入细胞中。当外泌体在细胞内破裂并且基因或其表达产物的抑制剂(图1中的抗miRNA核酸)被释放时,基因或其表达产物的功能受到抑制。当待被抑制功能的基因或其表达产物参与癌症增殖或转移时,本发明的缀合物用作癌症增殖和/或转移抑制剂。对于成人来说,本发明的缀合物可以以每天约1μg至1g的剂量施用,用于抑制癌症增殖和/或转移。
实施例
下面参考实施例更具体地解释本发明。然而,本发明不限于这些实施例。
在实施例中,使用由Cosmo Bio Co.,Ltd.制备的抗CD63抗体、由abcam制备的抗CD9抗体和抗CD81抗体、以及由abnova制备的抗TSG101抗体。
实施例1:将荧光标记的抗体捕获到细胞中(图2)
对识别外泌体表面抗原的抗体是否与外泌体一起掺入细胞中进行验证。
将Hela细胞(宫颈癌细胞)以9000/孔的量接种到多孔玻璃底皿(Matsunami GlassInd.,Ltd)上,并使用5%CO2孵化器在37℃下孵育24小时。向每个孔中加入荧光标记的抗体(抗CD63抗体、抗CD9抗体、抗CD81抗体和抗TSG101抗体),然后使用5%CO2孵化器在37℃孵育24小时。除去上清液,以1×PBS洗涤细胞。加入100μL的4%多聚甲醛,然后在室温下孵育5分钟,从而固定细胞。用1×PBS洗涤细胞两次。加入在1×PBS中的200μL的Hoechst33342(最终浓度=5μM),然后在室温下孵育10分钟,从而对活细胞染色。用1×PBS洗涤细胞两次,并进行共聚焦激光显微镜成像。图3示出了结果。如图3所示,在用抗CD63抗体(CD63)处理的细胞中,清楚地观察到作为抗体标记基团的荧光素的荧光。对于抗CD9抗体(CD9)、抗CD81抗体(CD81),略微观察到荧光素的荧光。当使用抗TSG101抗体(TSG101)时,未观察到荧光素的荧光。
尽管在使用Hela细胞的上述实施例中掺入了抗CD63抗体,但当使用Cal27细胞代替Hela细胞时,优先掺入抗CD9抗体。
可以认为,抗CD63抗体和抗CD9抗体首先结合到细胞培养上清液中存在的外泌体,然后掺入细胞中。
实施例2和比较例1:将抗体/核酸缀合物捕获到细胞中(图4)
对识别外泌体表面抗原的抗体是否与外泌体一起掺入细胞中进行验证。
将Hela细胞(宫颈癌细胞)以9000/孔的量接种到多孔玻璃底皿(Matsunami GlassInd.,Ltd)上,并使用5%CO2孵化器在37℃下孵育24小时。向每个孔中加入抗CD63抗体-9r/核酸缀合物(抗CD63IgG-9r+抗miR(Cy5))。本文使用的抗miR(Cy5)是5'-Cy5-aguca auagggugug ugaga gacuu acug-3'(FASMAC,SEQ ID NO:1)。
作为比较例1,加入抗CD63IgG和抗miR(Cy5)代替抗CD63IgG-9r/核酸缀合物。
鬼笔环肽用于细胞骨架的染色。鬼笔环肽是一种寡肽,其特异性结合至构成细胞骨架的聚合肌动蛋白(F-肌动蛋白)。
使用5%CO2孵化器在37℃下孵育24小时。除去上清液,用1×PBS洗涤细胞。加入100μL的4%多聚甲醛,然后在室温下孵育5分钟,从而固定细胞。用1×PBS洗涤细胞两次,加入在1×PBS中的200μL的Alexa488-标记的鬼笔环肽溶液(终浓度=100nM),然后在室温下孵育20分钟。除去鬼笔环肽溶液,加入在1×PBS中的200μL的Hoechst33342(最终浓度=5μM),然后在室温下孵育10分钟,从而对活细胞染色。用1×PBS洗涤细胞两次,并进行共聚焦激光显微镜成像。图5示出了结果。如图5所示,示出了抗CD63抗体/核酸缀合物(抗CD63IgG-9r+抗miR(Cy5))被掺入了细胞中。
实施例3:抗CD63抗体/抗miR核酸缀合物的微小RNA功能抑制作用(图6)
评估了在将抗CD63抗体/抗miR核酸缀合物掺入细胞后发挥微小RNA功能抑制作用。
将Hela细胞(宫颈癌细胞)以4500/孔的量接种到96孔板上,并使用5%CO2孵化器在37℃下孵育24小时。将目标荧光素酶mRNA的微小RNA(miR-Luc)导入各孔(LipofectamineRNAiMAX)。使用5%CO2孵化器在37℃下孵育18小时,并引入表达荧光素酶的质粒(pGL4.13和pGL4.73)(Lipofectamine 2000)。使用5%CO2孵化器在37℃下孵育6小时,并且抗CD63抗体/抗miR核酸缀合物(抗CD63IgG-9r+抗miR-Luc),仅加入抗miR-Luc(300nM)或仅加入抗CD63抗体(600nM),并使用5%CO2孵化器在37℃下孵育24小时;然后加入萤火虫荧光素并进行荧光素酶测定。缀合物是抗CD63抗体(300nM)/抗miR核酸(300nM)、或抗CD63抗体(600nM)/抗miR核酸(300nM)。图7示出了结果。
证实抗-CD63抗体/抗miR核酸缀合物在缀合物掺入细胞后发挥微小RNA功能抑制作用。
实施例4:抗CD63抗体/抗miR核酸缀合物对外泌体包封的微小RNA功能抑制作用
评估了抗CD63抗体/抗miR核酸缀合物是否发挥外泌体包封的微小RNA功能抑制作用。
将Cal27(口腔上皮癌细胞)以50000/孔的量接种到96孔板上,并使用5%CO2孵化器在37℃下孵育24小时。刮下细胞,然后在缺氧(0.1%O2)或常氧(20%O2)下培养;之后,加入外泌体(10μg/ml)。随后,在缺氧外泌体处理的系统中,用抗CD63抗体/抗miR核酸缀合物(抗CD63IgG-9r+抗miR21)、抗CD63抗体+抗miR-21(无连接基团)进行孵育、或使用5%CO2孵化器在37℃下无添加的系统(对照)培养24小时。此后,观察到划痕伤口闭合(伤口闭合%)。图8示出了结果。
证实了抗CD63抗体/抗miR核酸缀合物抑制外泌体miR21依赖性细胞增殖。
实施例5:抗CD63抗体/抗miR 21核酸缀合物在体内的功能抑制作用(图9)
评估了抗CD63抗体/抗miR核酸缀合物是否也在体内发挥微小RNA功能抑制作用。
将Cal27(口腔上皮癌细胞)以500000/200μL/PBS的量移植到裸鼠的臀部区域(nu/nu BALB)。14天后,测量肿瘤系统。通过仅将PBS与Cal27一起给药来制备对照(PBS);将对照与抗CD63抗体/抗miR21核酸缀合物(抗CD63IgG-9r+抗miR21)或抗CD63抗体+抗miR21给药组(每组单独给药)进行比较。图9示出了结果。
证实了本发明的缀合物在体内具有肿瘤增殖抑制作用。
序列表
<110> 国立大学法人京都大学
<110> 学校法人京都药科大学
<120> 能够抑制外泌体中的遗传功能的复合物以及癌症增殖和/或转移抑制剂
<130> P16-213WO
<150> JP 2016-028924
<151> 2016-02-18
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<170> PatentIn 版本 3.5
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<212> RNA
<213> 人工序列
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<223> 反义 RNA
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<213> 人工序列
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<223> 聚精氨酸
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Claims (12)
1.一种缀合物,包含靶向外泌体表面抗原的抗体或抗体片段,以及基因或其表达产物的抑制剂,其中所述抗体或抗体片段和所述基因或其表达产物的抑制剂直接或通过连接基团共价结合或非共价结合。
2.根据权利要求1所述的缀合物,其中所述外泌体表面抗原是CD9、CD63、CD81或CD147。
3.根据权利要求1或2所述的缀合物,其中所述抗体或抗体片段采用含有选自由半胱氨酸、精氨酸、赖氨酸和鸟氨酸组成的组中的至少一种氨基酸的肽进行修饰,并且所述基因或其表达产物的抑制剂通过共价键(S-S键)、配位键或非共价键与肽结合。
4.根据权利要求3所述的缀合物,其中所述肽还包含甘氨酸或丙氨酸。
5.根据权利要求3所述的缀合物,其中所述肽是聚赖氨酸或聚精氨酸。
6.根据权利要求5所述的缀合物,其中所述肽是聚精氨酸。
7.根据权利要求1至6中任一项所述的缀合物,其中所述基因或其表达产物的抑制剂是抗miRNA核酸,并且所述抗miRNA核酸是通过与所述外泌体中含有的miRNA形成互补链而抑制miRNA功能的核酸。
8.根据权利要求1所述的缀合物,其中所述抗体或抗体片段是抗CD63抗体。
9.根据权利要求1的缀合物,其中所述基因或其表达产物的抑制剂是所述外泌体中含有的miRNA或基因的抑制剂。
10.根据权利要求1所述的缀合物,其中所述基因或其表达产物的抑制剂是miRNA抑制剂。
11.根据权利要求1所述的缀合物,其中所述抗体或抗体片段是单克隆抗体、单链抗体、Fab、Fab'、F(ab')2、Fv或scFv。
12.一种癌症增殖和/或转移抑制剂,包含根据权利要求1-11中任一项所述的缀合物。
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