CN107033216A - 靶向癌干细胞的胜肽和其应用 - Google Patents
靶向癌干细胞的胜肽和其应用 Download PDFInfo
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Abstract
本发明公开靶向癌干细胞的胜肽和其应用,具体涉及一种靶向癌干细胞的合成的胜肽。所述胜肽由SEQ ID NO:1至SEQ ID NO:15任一的氨基酸序列所组成。本发明还公开一种组合物,包含所述的合成的胜肽,其具有接合至其上的一治疗剂,以及一医药上可接受的载剂或稀释剂。本发明还公开一种筛选特异性结合至癌干细胞的胜肽的方法,包含藉由使用一噬菌体表达系统来建立一寡胜肽库,使该寡胜肽库中的噬菌体与一癌细胞系的一般癌细胞的培养物接触,使未与所述一般癌细胞结合的噬菌体与该癌细胞系的癌干细胞的培养物接触,以及从与所述癌干细胞结合的噬菌体中筛选出特异性结合至癌干细胞的胜肽。
Description
技术领域
本发明涉及一种靶向癌干细胞的合成的胜肽。本发明还公开了包含该装置的套组。本发明还公开一种组合物,包含所述的合成的胜肽。本发明还公开一种筛选特异性结合至癌干细胞的胜肽的方法。
背景技术
自1994年首次发现急性骨髓性白血病的癌干细胞(CSCs)以来,亦在乳癌、脑癌、肝癌、结肠癌、肺癌和胰脏癌中证实分辨出CSCs。除了一般干细胞的自我更新活性,CSCs具有对化疗、放疗、低氧及免疫监测的抗性。此外,CSCs被认为是癌症复发的主因。
仍需要特异性靶向癌干细胞的药剂,以用于治疗、影像和其他目的。
发明内容
在一方面,本发明提供一种靶向癌干细胞的合成的胜肽,其由SEQ ID NO:1至SEQID NO:15任一的氨基酸序列所组成。
另一方面,本发明提供所述的合成的胜肽在癌症的诊断的应用。
又一方面,本发明提供所述的合成的胜肽在将治疗剂靶向癌干细胞的应用。
所述的合成的胜肽可接合至一可侦测部分(detectable moiety)。在本发明的部分具体实施例中,所述可侦测部分为一放射性标记、一荧光团或一磁性成像造影剂。
所述的合成的胜肽可接合至一治疗剂。在本发明的部分较佳具体实施例中,所述治疗剂为一抗癌剂。所述抗癌剂包括但不限于一化疗剂、一放射治疗剂或一免疫治疗剂。
另一方面,本发明提供一种组合物,其包含所述的合成的胜肽以及一医药上可接受的载剂或稀释剂。较佳地,所述的合成的胜肽结合至一治疗剂。
又另一方面,本发明提供一种筛选特异性结合至癌干细胞的胜肽的方法,其包含:藉由使用一噬菌体表达系统来建立一寡胜肽库;使该寡胜肽库中的噬菌体与一癌细胞系的一般癌细胞的培养物接触;使未与所述一般癌细胞结合的噬菌体与该癌细胞系的癌干细胞的培养物接触;以及从与所述癌干细胞结合的噬菌体中筛选出特异性结合至癌干细胞的胜肽。
从以下的配合附图的较佳具体实施例的说明,了解本发明的所述方面及其他方面,然而,在不悖离本文揭露的创新概念的精神与范围下可进行变化及修改。
附图说明
图1A显示癌干细胞(CSC)归巢胜肽(HPs)对CSCs的选择性结合。经罗丹明B标记的CSC HP1(Rd-CSC HP-1)和CSC HP2(Rd-CSC HP-2)以及经FITC记的CSC HP11(FITC-CSC HP-11)被直接用来对EMT6CSCs和癌细胞(CCs)染色。细胞以DAPI复染。在相同的曝光条件下撷取荧光影像。
图1B显示CSC HPs对CSCs的选择性结合。EMT-6、CT26、Hepa1-6和PANC-1的CSCs分别以Rd-CSC HP-1、Rd-CSC HP-2、FITC-CSC HP-3、FITC-CSC HP-8、FITC-CSC HP-9、FITC-CSC HP-10或FITC-CSC HP-11染色。在相同的曝光条件下撷取荧光影像。
图2显示CSC HP-hP1-DsRed对PANC-1CCs和CSCs的结合。最终浓度为25μg/mL的CSCHP-hP1-DsRed重组蛋白被用来对PANC-1CCs和CSCs染色。箭头指出在PANC-1CC组别中观察到的微弱信号。
图3A-图3F显示透过聚糖微阵列和免疫荧光分析的CSC HPs的靶标)。图3A显示以生物素标记的CSC HP-hP1的聚糖微阵列分析结果。图3B显示以生物素标记的CSC HP-9的聚糖微阵列分析结果。图3C显示Globo系列聚糖(聚糖26)对Lacto/Neolacto系列聚糖(glycan19)的相对信号强度,其以同一微阵列的收集自15个CSC HPs的阳性对照组信号3进行归一化(normalized)。图3D显示来自15个CSC HPs的聚糖63和聚糖69的相对信号强度。图3E显示收集自CSC HP-10、CSC HP-11和CSC HP-hP2芯片的聚糖66(Gal-b-1,3-GalNAc-b-)、聚糖67(Gal-b-1,3-(Neu5Ac-a-2,6)-GalNAc-b-)、聚糖68(Neu5Ac-a-2,6-Gal-b-1,3-GalNAc-b-)、聚糖69(Neu5Ac-a-2,6-Gal-b-1,3-(Neu5Ac-a-2,6)-GalNAc-b-)和聚糖70(Neu5Ac-a-2,3-Gal-b-1,3-(Neu5Ac-a-2,6)-GalNAc-b-)信号。图3F为PANC-1CSCs上的干细胞特异性聚糖标记的免疫荧光影像,其以抗Globo系列(SSEA-4和GbH)、抗Lacto/Neolacto(Lc/nLc)系列(SSEA-1and SSEA-5)和抗Lewis Y的单克隆抗体来完成。
图4A为一流程图,显示自原始的M13PhD7库的第一级筛选,以制备EMT6CSC HP库,以及第二级筛选以产生CT26CSC HP库、Hepa1-6CSC HP库、CSC/ESC HP库和CSC HP库。
图4B为一流程图,例示PANC-1CSC HP库的建立。
具体实施方式
除非另有定义,本文所使用的所有技术和科学术语,具有与本发明所属技术领域中具有通常知识者所理解的相同的涵义。
在一方面,本发明提供一种靶向癌干细胞的合成的胜肽,其由SEQ ID NO:1至SEQID NO:15任一的氨基酸序列所组成。所述合成的胜肽能够选择性地或特异性地归巢至(homes to)癌干细胞,因此其可以一结合物的形式使用,以将透过全身性投药的治疗剂选择性地靶向至癌干细胞。这样的剂选择性靶向能够增加投递至癌干细胞的药剂的有效量,同时减低药剂对其他细胞或身体的其他部分造成副作用的可能性。
在本文中所使用的术语“癌干细胞(cancer stem cells,CSCs)”是指哺乳动物的癌干细胞,例如,小鼠的或人类的癌干细胞。
本发明的合成的胜肽归巢至、靶向至或特异性地结合至CSCs,但不与(一般)癌细胞结合或非常弱地结合至(一般)癌细胞。所述癌干细胞包括但不限于以下癌症的癌干细胞:乳癌、肝癌、胰脏癌及结肠癌(大肠癌)。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列SQPTWMF(SEQ ID NO:1)所组成(亦称为“CSC HP-1”)。在一具体实施例中,CSC HP-1归巢至、靶向至或特异性地结合至乳癌的CSCs。在另一具体实施例中,CSC HP-1归巢至、靶向至或特异性地结合至结肠癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列GMMSSPP(SEQ ID NO:2)所组成(亦称为“CSC HP-2”)。在一具体实施例中,CSC HP-2归巢至、靶向至或特异性地结合至乳癌的CSCs。在另一具体实施例中,CSC HP-2归巢至、靶向至或特异性地结合至结肠癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列FSGGGNH(SEQ ID NO:3)所组成(亦称为“CSC HP-3”)。在一具体实施例中,CSC HP-3归巢至、靶向至或特异性地结合至乳癌的CSCs。在另一具体实施例中,CSC HP-3归巢至、靶向至或特异性地结合至结肠癌的CSCs。在另一具体实施例中,CSC HP-3归巢至、靶向至或特异性地结合至肝癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列FPFTKNL(SEQ ID NO:4)所组成(亦称为“CSC HP-4”)。在一具体实施例中,CSC HP-4归巢至、靶向至或特异性地结合至乳癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列ATYGNLW(SEQ ID NO:5)所组成(亦称为“CSC HP-5”)。在一具体实施例中,CSC HP-5归巢至、靶向至或特异性地结合至乳癌的CSCs。在另一具体实施例中,CSC HP-5归巢至、靶向至或特异性地结合至结肠癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列YHMPALM(SEQ ID NO:6)所组成(亦称为“CSC HP-6”)。在一具体实施例中,CSC HP-6归巢至、靶向至或特异性地结合至乳癌的CSCs。在另一具体实施例中,CSC HP-6归巢至、靶向至或特异性地结合至结肠癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列HGGVRLY(SEQ ID NO:7)所组成(亦称为“CSC HP-7”)。在一具体实施例中,CSC HP-7归巢至、靶向至或特异性地结合至乳癌的CSCs。在另一具体实施例中,CSC HP-7归巢至、靶向至或特异性地结合至结肠癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列ELTPLTL(SEQ ID NO:8)所组成(亦称为“CSC HP-8”)。在一具体实施例中,CSC HP-8归巢至、靶向至或特异性地结合至乳癌的CSCs。在另一具体实施例中,CSC HP-8归巢至、靶向至或特异性地结合至结肠癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列GPSASRN(SEQ ID NO:9)所组成(亦称为“CSC HP-10”)。在一具体实施例中,CSC HP-10归巢至、靶向至或特异性地结合至乳癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列GLAPFNA(SEQ ID NO:10)所组成(亦称为“CSC HP-11”)。在一具体实施例中,CSC HP-6归巢至、靶向至或特异性地结合至乳癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列KIYTTLD(SEQ ID NO:11)所组成(亦称为“CSC HP-9”)。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列NLQPPAY(SEQ ID NO:12)所组成(亦称为“CSC HP-12”)。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列GPKVTIW(SEQ ID NO:13)所组成(亦称为“CSC HP-Hp1”)。在一具体实施例中,CSC HP-Hp1归巢至、靶向至或特异性地结合至胰脏癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列GSPVMSW(SEQ ID NO:14)所组成(亦称为“CSC HP-Hp2”)。在一具体实施例中,CSC HP-Hp2归巢至、靶向至或特异性地结合至胰脏癌的CSCs。
根据本发明的一具体实施例,所述合成的胜肽由氨基酸序列YHQVKPH(SEQ ID NO:15)所组成(亦称为“CSC HP-Hp3”)。在一具体实施例中,CSC HP-Hp3归巢至、靶向至或特异性地结合至胰脏癌的CSCs。
本发明的合成的胜肽可透过常规方法(例如,化学合成)来制备或合成。
根据本发明的部分具体实施例,所述合成的胜肽用于癌症的诊断。根据本发明的一些其他具体实施例,所述合成的胜肽用于将治疗剂靶向癌干细胞。
根据本发明,所述合成的胜肽接合至(fused to)一可侦测部分(detectablemoiety)。在本发明的部分具体实施例中,所述可侦测部分为一放射性标记、一荧光团或一磁性成像造影剂。
根据本发明,所述的合成的胜肽可接合至一治疗剂。在本发明的部分较佳具体实施例中,所述治疗剂为一抗癌剂。所述抗癌剂包括但不限于一化疗剂、一放射治疗剂或一免疫治疗剂。本文中所使用的术语“免疫治疗剂(immunotherapeutic agent)”是指一药剂,其能够在治疗上增进或抑制以该药剂处理的个体的免疫系统。
上述的可侦测部分或治疗剂可直接地或间接地结合至或接合至一本发明的合成的胜肽,例如,透过共价键或离子键结合。
另一方面,本发明提供一种组合物,其包含所述的合成的胜肽以及一医药上可接受的载剂或稀释剂。较佳地,所述的合成的胜肽结合至一治疗剂。所述组合物可作为一医药组合物使用。所述组合物可透过混合所述合成的胜肽和一医药上可接受的载剂或稀释剂来制备,或透过将一医药上可接受的载剂或稀释剂化学地结合至所述合成的胜肽来制备。
本发明的合成的胜肽或组合物可透过静脉内给药来投药给有需要的个体。
又另一方面,本发明提供一种筛选特异性结合至癌干细胞的胜肽的方法,其包含:藉由使用一噬菌体表达系统来建立一寡胜肽库;使该寡胜肽库中的噬菌体与一癌细胞系的一般癌细胞的培养物接触;使未与所述一般癌细胞结合的噬菌体与该癌细胞系的癌干细胞或癌干细胞样细胞(cancer stem cell like cells)的培养物接触;以及从与所述癌干细胞结合的噬菌体中筛选出特异性结合至癌干细胞的胜肽。
根据本发明的较佳具体实施例,所述癌干细胞或癌干细胞样细胞源自或得自一瘤球(tumorosphere)(Weiswald et al.,Neoplasia,17(1):1-15(2015)),该瘤球制备自一般的癌细胞或肿瘤细胞。所述瘤球可透过一无血清培养方法来制备。例如,Eramo et al.,Cell Death Differentiation,15:504-514(2008)、Cioce et al.,Cell Cycle,9:2878-2887(2010);Cao et al.,BMC Gastroenterol.,11:71(2011)或Chen et al.,Clin.Exp.Metastasis,28(8):751-763(2011))中所公开的方法。
在现有技术中,大部分的注意力放在靶向(一般)癌细胞的胜肽或其他实体,部分是因为透过细胞培养得到的癌干细胞的量很低。此外,就发明人所知,从未有人提出筛选不结合至一般癌细胞但结合至癌干细胞的胜肽,这样的筛选策略。然而,发明人非预期性地发现,透过上述的方法以及使用源自瘤球的癌干细胞或癌干细胞样细胞,能够有效地筛选出特异性结合至癌干细胞的胜肽。
透过以下的非限制性实例来进一步说明本发明。
实例
材料及方法
1.癌细胞和癌干细胞的培养
养虽然CSCs可以透过诸如CD44和CD133的表面标志来富集,能够选择性地增殖CSCs的实用且简单的方法,对研究而言非常关键。在2008年发展出能够选择性地增殖人类肺CSCs的开创性的无血清培养方法(Eramo et al.,Cell Death Differentiation,15:504-514(2008))。自那时候起,乳癌(Cioce et al.,Cell Cycle,9:2878-2887(2010))、肝癌(Cao et al.,BMC Gastroenterol.,11:71(2011))和结肠癌(Chen et al.,Clin.Exp.Metastasis,28(8):751-763(2011))的CSCs亦被报导。CSCs以球形的聚合形状生长,研究者将其命名为瘤球(Weiswald et al.,Neoplasia,17(1):1-15(2015))。
重组的生长因子购自PeproTech。小鼠的乳癌EMT6细胞系、肝癌Hepa1-6细胞系和人类胰腺导管腺瘤(pancreatic ductal adenoma)细胞系于补充有10%FCS的DMEM中维持。小鼠的结肠癌The mouse colon cancer CT26细胞系于补充有10%FCS的RPMI1640中培养。用于CSCs的基础培养基为含有2mM谷氨酰胺的DMEM/F12,EMT6CSCs的补充物为25ng/Ml的rmEGF、25ng/mL的rmFGF2、5μg/mL的胰岛素、4μg/mL的肝素、0.5μg/mL的氢化可的松和1%的BSA;CT26的补充物为20ng/mL rmEGF、5μg/mL的胰岛素、2%的B27和0.4%BSA;Hepa1-6CSCs的补充物为20ng/mL的mEGF、10ng/mL的rmFGF2、2%的B27和1%的N2;PANC-1CSCs的补充物为2%的B27和20ng/mL的rhFGF2。为了制备瘤球,在无血清培养基中以低密度(例如,小于104细胞/mL)。培养7天后,以40μm细胞过滤器收获瘤球,并于室温以900×g离心5分钟。透过胰蛋白酶将瘤球的团粒分离为多个单一细胞,然后将所得到的细胞再次扩增为瘤球,为期7天。每隔一天补充生长因子,分离自第三至五轮扩增的瘤球用于实验。
2.M13噬菌体展示
含有109独立克隆的M13PhD-7库(NEB,E8100)购自新英格兰生物实验室公司(NewEngland Biolabs Inc)。在T75烧瓶中,10mL DMEM中的1013斑块固结单位(pfus)的M13PhD-7噬菌体以癌细胞预吸附2次,每次1小时。接着,将上清液移至含有5×106CSCs的T25烧瓶中,培养1小时。透过在DMEM/0/2%BSA中离心和悬浮3次,以去除未结合或弱联结的噬菌体。将细胞团粒悬浮于含有109E.coli ER2738的1mL PBS中,并在振荡器中培养1小时。之后加入10mL LB/5mM MgCl2。于37℃过夜培养后,透过离心去除细胞,并滴定上清液中的噬菌体。针对EMT6和PANC-1,进行上述的筛选程序三轮,来分别获得第一级EMT6CSC HP库和PANC-1CSCHP库。然后,挑出M13斑块以制备用于定序的DNA。所述级EMT6CSC HP库和PANC-1CSC HP库进一步以CT26CSCs、Hepa1-6CSCs和小鼠胚胎干细胞(mESC)筛选,来制备第二级CT26CSC HP库、Hepa1-6CSC HP库和CSC/ES HP库。收集mESC吸附后未结合的部分,命名为CSC PH库。参见图4A和图4B。
3.CSC HPs对CSCs的结合
20μg/mL的以罗丹明B标记的CSC HP1(Rd-CSC HP-1)和CSC HP2(Rd-CSC HP-2),以及以FITC标记的CSC HP11(FITC-CSC HP-11)被直接用来对EMT6CSCs和癌细胞(CCs)染色。细胞以DAPI复染。
4.聚糖微数组分析
具有下表A中所列序列的N端结合有生物素的CSC HPs通过GeneDirex以高于95%的纯度合成。将生物素-CSC HP溶解于DMSO中以制备10mg/mL储备液。
表A.以生物素标记的CSC HPs
聚糖阵列芯片100(Cat.#GA-Glycan-100)购自RayBiotech,并依制造商的指示处理(稍加修饰)。简言之,在室温下用400μL样品稀释液(Item E)阻断30分钟后,将结合有生物素的CSC HPs用样品稀释剂稀释至1μg/400μL,并在室温下进行杂交反应2小时。洗涤后,向每个孔中加入400μL的1×Cy3-共轭的链霉亲和素。培养室用塑料粘合条覆盖并且用铝箔覆盖载玻片以避免在暗室中培养期间受光照。将载玻片与CY3-共轭的链霉亲和素在室温下培养1小时,伴随温和晃动或摇动。彻底洗涤后,将载玻片完全排干,并用激光扫描仪AxonGenePix读取其上的荧光信号。将聚糖X的相对强度定义为(X–阴性对照的平均值)/(阳性对照–阴性对照)×100%。
5.免疫荧光(IF)分析
初级抗体兔多克隆抗CD44(GTX102111)、抗E-钙粘着蛋白(GTX61823)和抗CDSN(GTX110093)以及小鼠单克隆抗SSEA-1(IgM,GTX48038)、抗SSEA-5(IgG,GTX70019)和抗Lewis Y(IgM,GTX75903)购自GeneTex;小鼠单克隆抗SSEA-4(IgG,90230)、抗TRA-1-60和TRA-1-81(IgM,在90232中)来自Millipore;小鼠单克隆抗GbH(IgM,ALX-804-550)来自Enzo。第二抗体DyLight594-共轭的山羊抗兔IgG(111-515-144)和DyLight488-共轭的驴抗小鼠IgG(715-485-151)获自Jackson Lab。DyLight594-共轭的山羊抗小鼠IgM(GTX76754)获自GeneTex。用4%多聚甲醛/PBS固定癌细胞和瘤球,并用0.5%NP-40/1%BSA/PBS(PBSNB)透化(permeabilized)。然后将细胞与在PBSNB中稀释50-100倍的一抗培养2小时。用0.05%NP-40/PBS(PBSN)洗涤4次后,将细胞与二抗(在PBSNB中1/200稀释)一起培养2小时。以PBSN洗涤4次和用水短暂洗涤1次后,用DAPI固定细胞。
实例1:小鼠EMT6、CT26和Hepa1-6CSCs的鉴定
挑选由M13噬菌体展示的胜肽是否专一于癌干细胞的标准取决于CSCs和癌细胞(CC)之间的表面特征的差异。通过检查特异性干细胞表面标记来鉴定制备为肿瘤球形式中的小鼠EMT6、CT26和Hepa1-6CSCs(数据未显示)。干细胞表面标记TRA-1-60高度表达在所有三株CSCs,但其在对应的CC的表达是低的或检测不到的,而组成型表达的(constitutivelyexpressed)表面蛋白角质软蛋白(CDSN)在CSCs和CCs都有检测到(数据未显示)。除了显示在EMT6CSCs上弱表达的SSEA-4之外,所有的人类CSC通用标记物:CD44和E-钙粘蛋白,以及多能干细胞标记物SSEA-1、SSEA-5、TRA-1-60和TRA-1-81,都证实在所有三株小鼠CSC上有强的表达(数据未显示)。
实例2:第一级EMT6CSC HP库
为了研究CSC特异性归巢胜肽(HP),M13PhD7七胜肽库以EMT6CCs预吸附以除去一般的癌症HP,然后将游离的噬菌体转移到EMT6CSC用于正选择。获取联结在EMT6CSC上的噬菌体并以大肠杆菌ER2738扩增。上述筛选程序进行三轮,以制备富集的初级EMT6CSC HP库。随机挑取独立的噬菌斑,读取31个有效序列并整理于下表1中。
表1:在第一级和第二级CSC HP库中检测到CSC HP序列的次数
胜肽SQPTWMF(称为CSC HP-1)(SEQ ID NO:1)、GMMSSPP(称为CSC HP-2)(SEQ IDNO:2)、FSGGGNH(称为CSC HP-3)(SEQ ID NO:3)、FPFTKNL(称为CSC HP-4)(SEQ ID NO:4)和ATYGNLW(称为CSC HP-5)(SEQ ID NO:5)分别出现10、6、5、3和2次。胜肽YHMPALM(称为CSCHP-6)(SEQ ID NO:6)、HGGVRLY(称为CSC HP-7)(SEQ ID NO:7)、ELTPLTL(称为CSC HP-8)(SEQ ID NO:8)、GPSASRN(称为CSC HP-10)(SEQ ID NO:9)和GLAPFNA(称为CSC HP-11)(SEQID NO:10)各出现一次。由于M13PhD7库估计含有109个独立克隆,因此可以得出结论,筛选方法是高度有效的。
实例3:第二级CT26CSC HP库、Hepa1-6CSC HP库、CSC/ESC HP库和CSC HP库
通过CT26或Hepa1-6CSC对第一级CSC HP文库直接筛选择一次,以测试CSC HP对不同种类癌症的亲和力的偏差。如表1所示,CSC HP-8和CSC HP-3分别更特异于CT26和Hepa1-6CSC。CSC HP亲和力在CSC和胚胎干细胞(ESC)之间的偏差是另一个有趣的发现。CSC HP-1在ESC结合级分(CSC/ESC-HP库)中被进一步富集。在ESC未结合级分(CSC HP-文库)中读取到两个新的胜肽序列KIYTTLD(CSC HP-9)(SEQ ID NO:11)和NLQPPAY(CSC HP-12)(SEQ IDNO:12)(见表1)。
实例4:CSC HP和CSCs之间的相互作用
根据如上述的结果,选择CSC HP-1、CSC HP-2、CSC HP-3、CSC HP-8、CSC HP-9、CSCHP-10和CSC HP-11与罗丹明B或荧光素共轭(缀合)。所有这些肽都与EMT6、CT26和Hepa1-6CSCs有特异性的关联(结合)。它们在平行CC实验中未被检测到(图1A和图1B)。想要确定的是,这些源自小鼠癌症的CSC HP是否可识别人类CSC。发现从人胰腺癌细胞系PANC-1制备的瘤球确实会与上述七个胜肽关联(结合)(图1B)。
实例5:第一级PANC-1CSC HP库
如果由小鼠CSC筛选的CSC HP可以与人类CSC交叉反应,人类CSC筛选的CSC HP是否与小鼠CSC相关联?按照用于EMT6CSC HP筛选的类似方法,以人胰腺PANC-1CC进行三轮阴性选择和用PANC-1CSC阳性选择后,建立初级PANC-1CSC HP库。随机挑取独立斑块,读取20个有效序列。胜肽GPKVTIW(称为CSC HP-hP1),GSPVMSW(称为CSC HP-hP2)和YHQVKPH(称为CSC HP-hP3)分别出现17、2和1次。参见下表2。
表2.在第一级PANC-1CSC HP库中检测到CSC HP序列的次数
CSC HP-hP系列的氨基酸序列与小鼠CSC HP的氨基酸序列不匹配。除了化学合成的FITC-CSC HP-hP1(其可以选择性地与人和小鼠CSC结合)(数据未显示)之外,制备CSCHP-hP1-DsRed重组蛋白以测试CSC HP-hP1和PANC-1CSCs之间的相互作用是否受到接合至CSC HP-hP1胜肽C端的大分子载体的影响。证实CSC HP-hP1-DsRed重组蛋白能够选择性地结合至PANC-1CSCs,而CC内的一些细胞也被重组蛋白染色,如图2中的箭头所示。
实例6:CSC HP的靶标
开发出的CSC HP可以区分CSC和CC,并且发现这种现象在人和小鼠之间是跨种的。因此,进行实验来找出这些CSC HP的靶标。没有残留在硝酸纤维素膜上的FITC-CSC HP的可检测的荧光信号,所述硝酸纤维素膜已经通过SDS PAGE分离的CSC膜蛋白印迹(数据未显示)。使用其上点样4次100种寡糖的聚糖微阵列(聚糖阵列100微芯片)来分析寡糖和CSC HP之间的相互作用的可能性。表2中列出的生物素-CSC HP与芯片上的聚糖杂交。用Cy3-共轭的链霉亲和素探测在芯片上捕获的生物素-CSC HP,并通过激光扫描仪读取其上的荧光信号。图3A-3F中显示两个主要的模式。第一个包括Globo系列的聚糖16(Gal-β-1,4-Glc-β-)、18(Gal-α-1,4-Gal-β-1,4-Glc-β-)和26(GalNAc-β-1,3-Gal-α-1,4-Gal-β-1,4-Glc-β-)图3A)。典型的例子是CSC HP-1和CHC HP-hP1(图3C)。除了Globo系列的信号,第二个主要的模式涉及聚糖19(GlcNAc-β-1,3-Gal-β-1,4-Glc-β-,其为Lacto/Isolacto三糖骨架),其类似于信号26的水平(图3B)。典型的例子是CSC HP5、CSC HP6、CSC HP7和CSC HP9(图3C)。还侦测到两个与肿瘤恶性和转移有关的聚糖标记:聚糖63(Fuc-α-1,2-Gal-β-1,4-(Fuc-α-1,3)-GlcNAc-β-,亦称为Lewis Y表位)和聚糖69(Neu5Ac-α-2,6-Gal-β-1,3-(Neu5Ac-α-2,6)-GalNAc-β-)。CSC HP-1和CSC HP-hP1在聚糖63上展现较高的信号,而CSC HP-9、CSC HP-10、CSC HP-11、CSC HP-hP2和CSC HP-hP3在聚糖69上展现较高的信号(图3D)。两个Neu5Ac-α-2,6-残基都是CSC HP结合所必需的(图3E)。根据上述结果,制备人胰腺癌细胞PANC-1的CSCs,并且通过IF测定证实了在PANC-1CSC上有Globo系列的SSEA-4和GbH、Lacto/Isolacto系列的SSEA-1和SSEA-5以及Lewis Y等CSC聚糖标记(图3F)。
根据聚糖微阵列数据,CSC HP-1和CSC HP-hP1强且特异性地结合至聚糖26,其为四糖的Globo根结构。另一方面,CSC HP5、CSC HP6、CSC HP7和CSC HP9与聚糖19强烈结合,聚糖19是Lacto和Neolacto根结构四糖的核心三糖。发现被认为分别对肝和结肠CSC具有特异性的CSC HP-3和CSC HP-8与聚糖26弱结合。CSC HP-1和CSC HP-hP1与聚糖63(Lewis Y表位)有较强的结合。
本发明所属技术领域中具有通常知识者,基于本文所描述,在不需要进一步说明之下能够运用本发明至最广的程度。因此,应了解所提供的说明与权利要求范围仅为例示的目的,而非以任何方式限制本发明的范围。
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<400> 8
Glu Leu Thr Pro Leu Thr Leu
1 5
<210> 9
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 靶向癌干细胞的胜肽
<400> 9
Gly Pro Ser Ala Ser Arg Asn
1 5
<210> 10
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 靶向癌干细胞的胜肽
<400> 10
Gly Leu Ala Pro Phe Asn Ala
1 5
<210> 11
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 靶向癌干细胞的胜肽
<400> 11
Lys Ile Tyr Thr Thr Leu Asp
1 5
<210> 12
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 靶向癌干细胞的胜肽
<400> 12
Asn Leu Gln Pro Pro Ala Tyr
1 5
<210> 13
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 靶向癌干细胞的胜肽
<400> 13
Gly Pro Lys Val Thr Ile Trp
1 5
<210> 14
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 靶向癌干细胞的胜肽
<400> 14
Gly Ser Pro Val Met Ser Trp
1 5
<210> 15
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 靶向癌干细胞的胜肽
<400> 15
Tyr His Gln Val Lys Pro His
1 5
Claims (12)
1.一种靶向癌干细胞的合成的胜肽,其由SEQ ID NO:1至SEQ ID NO:15任一的氨基酸序列所组成。
2.如权利要求1所述的合成的胜肽在癌症的诊断的应用。
3.如权利要求1所述的合成的胜肽在将治疗剂靶向癌干细胞的应用。
4.根据权利要求1所述的合成的胜肽,其特征在于,其接合至一可侦测部分。
5.根据权利要求1所述的合成的胜肽,其特征在于,其接合至一治疗剂。
6.根据权利要求4所述的合成的胜肽,其中所述可侦测部分为一放射性标记、一荧光团或一磁性成像造影剂。
7.根据权利要求5所述的合成的胜肽,其中所述治疗剂为一抗癌剂。
8.根据权利要求7所述的合成的胜肽,其中所述抗癌剂为一化疗剂、一放射治疗剂或一免疫治疗剂。
9.一种组合物,其特征在于,其包含如权利要求5所述的合成的胜肽以及一医药上可接受的载剂或稀释剂。
10.一种筛选特异性结合至癌干细胞的胜肽的方法,其包含:
藉由使用一噬菌体表达系统来建立一寡胜肽库;
使该寡胜肽库中的噬菌体与一癌细胞系的一般癌细胞的培养物接触;
使未与所述一般癌细胞结合的噬菌体与该癌细胞系的癌干细胞的培养物接触;以及
从与所述癌干细胞结合的噬菌体中筛选出特异性结合至癌干细胞的胜肽。
11.根据权利要求10所述的方法,其特征在于,所述癌干细胞来自一瘤球(tumorosphere)。
12.根据权利要求11所述的方法,其特征在于,所述瘤球制备自一般癌细胞。
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