CN110621351A - 使用适配体的生物分子成像方法 - Google Patents
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- CN110621351A CN110621351A CN201880027916.2A CN201880027916A CN110621351A CN 110621351 A CN110621351 A CN 110621351A CN 201880027916 A CN201880027916 A CN 201880027916A CN 110621351 A CN110621351 A CN 110621351A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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Abstract
本发明涉及用于对肿瘤疾病区域成像的组合物,包括荧光标记或放射性同位素标记的ERBB2适配体,其中用放射性同位素或荧光染料标记的ERBB2适配体用于对肿瘤区域进行体内成像。
Description
技术领域
本发明涉及使用适配体的生物分子成像方法,并且更具体而言涉及通过使用用同位素标记的适配体并将其与表达人表皮生长因子受体2(HER2)的细胞系结合而获得图像的方法。
背景技术
适配体的词源来自拉丁语“aptus(完全正确)”和希腊语“meros(部分)”。适配体是具有由约20至90个碱基组成的DNA序列的单链核酸。通常,通过人工进化方法、例如体外SELEX(通过指数富集的配体的系统进化)作为适配体挖掘技术来筛选对靶分子具有高度特异性并对其具有高亲和力的适配体。因此,适配体被认为是非常合适的试剂,用于确定或发现被适配体靶向的特定分子的表达程度。在一些方面,例如降低的生产成本、易于合成、低毒性、不发生免疫应答、在动物系统中不产生与抗体不同的适配体等,与抗体相比,适配体具有优势。适配体是在诊断领域中相对较新开发的试剂。已经开发出针对各种靶标的许多适配体,包括针对凝血酶、核仁素、PSMA、TNC和病毒起源蛋白。在治疗领域,VEFG靶适配体被开发并于2004年被FDA批准用作老年性黄斑变性治疗剂。最近,在临床前和临床阶段正在开发如此多种类型的适配体,并且正在进行与诊断和治疗相关的大量实验。
HER2是本领域众所周知的癌症基因,其在约15至30%的乳腺癌中增加或过表达。此外,这是与不同癌症的高复发率和不良预后相关的因素。HER2激活了两个信号传导系统,包括促进细胞增殖的MAPK途径和增加癌细胞存活的PI3K-AKT途径。因此,上述因子是用于治疗癌症的高度优选的靶标。在这方面,用于靶向HER2的曲妥珠单抗和帕妥珠单抗目前作为本领域众所周知和可获得的治疗性单克隆抗体存在,并且已经发现在临床应用中是有效的。在此之前,通过传统的SELEX方法和基于细胞的SELEX公开了几种靶向HER2的DNA/RNA适配体。此外,最近已经报道了利用HER2适配体的抑癌性质的药学上的实例。
同时,分子图像可以是非侵入性的方法,其能够实时可视化关于活细胞或组织或物体的细胞分子水平的生化事件,而不会造成损害。可以将修饰为磁性纳米材料或荧光材料的适配体提供为用于靶向荧光成像或磁共振成像(MRI)的优选物质。一些体内MRI研究表明,在患有癌症的小鼠中可以有效地靶向癌症。但是,由于在解剖学改变之前发生了新陈代谢的变化,PET在诊断方面显然比解剖学技术(例如计算机断层扫描(CT)和MRI)更具优势。在临床应用中,PET广泛用于基础研究和临床前领域。例如,PET可以用于核实或验证新放射疗法的分析,新治疗剂的治疗功效以及药物的体内分布。PET的优点可能包括从临床前试验到临床试验的探针深度、出色的灵敏度、定量数据和可转换性(即阶段进展)。即,PET是可以检测活生物分子的目标水平中的生化变化并且具有高灵敏度的代表性分子成像装置,因此被广泛用于包括基础科学和临床前领域的应用。使用适配体靶向癌症是近年来提出的一种生物分子成像技术,例如,包括Hicke等人在内的许多研究者都已经在分子成像中采用了适配体。他们已经将99mTC结合到称为TTA1的适配体上,该适配体通过共价键与作为细胞外蛋白的腱生蛋白C结合,然后在体内使用伽马相机对癌症成像。从那时起,PET成像也被其他研究者团队实施。
然而,尚未公开使用HER2特异性ERBB2适配体的PET成像的实施。
发明内容
【技术问题】
适配体是核酸的一种并且是对靶分子具有高特异性和亲和力的材料。本发明的一个目的是提供使用放射性同位素或荧光染料标记的适配体的体内分子图像。
【技术方案】
图1是示出放射性同位素或荧光标记的ERBB2适配体的机理示意图。
根据本发明,用放射性同位素或荧光染料标记的HER2适配体用于体内成像。
【有益效果】
在流式细胞仪分析中,ERBB2适配体几乎不与不表达HER2的MDA-MB231细胞系结合,但对BT474作为表达HER2的细胞系具有很高的亲和力。类似地,从通过共聚焦显微镜获得的图像中观察到,适配体与表达HER2的乳腺癌细胞系结合,而仅显示与不表达HER2的细胞的最小结合。体内移植了BT474癌细胞系的小鼠的正电子发射断层扫描的分子图像已证明18F标记的HER2特异性ERBB2适配体的摄入量显著增加。ERBB2适配体可能在体内和体外都优先与表达HER2的乳腺癌细胞系结合,原因是在细胞表面上可能识别到HER2结构。
用放射性同位素(例如18F)或荧光染料标记的ERBB2适配体可以识别人乳腺癌细胞中HER2的表达并实现足够的可视化。这些结果提议了使用这样的同位素或荧光染料标记的ERBB2适配体对HER2阳性乳腺癌细胞的靶治疗应用或如何对其进行治疗的潜在应用方法。
附图说明
图1是示出放射性或荧光标记的ERBB2适配体的机理的示意图。
图2示出了使用Typhoon FLA 7000 3%琼脂糖凝胶分析R-[ERBB2适配体]-X-hy(bp)-Cy5的结果,R-[ERBB2适配体]-X-hy(bp)-Cy5是通过R-[ERBB2适配体]-ODN-X(R=H、胆固醇或PEG,并且X=H或idT)与cODN-Cy5杂交获得的产物。
图3示出了在50℃、55℃和60℃使用3%琼脂糖凝胶鉴定胆固醇-[AP001-24]-ODN-idT或胆固醇-[AP001-24]-ODN适配体和荧光标记的cODN(cODN-Cy5)之间的互补碱基配对的结果(黑色:适配体,红色:cODN-Cy5)。
图4示出使用琼脂糖凝胶、加热到95℃鉴定胆固醇-[AP001-24]-ODN-idT、胆固醇-[AP001-24]-ODN、PEG化的-[AP001-25]-ODN-idT或PEG化的-[AP001-25]-ODN适配体和荧光标记的cODN(cODN-Cy5)之间的互补碱基对的结果。
图5示出了用R-[ERBB2适配体]-X-hy(bp)-Cy5处理的KPL4、N87和SK-BR细胞系的共聚焦图像结果,包括HER2阳性细胞系中[AP001-24]-hy(bp)-Cy5和[AP001-25]-hy(bp)-Cy5适配体的共聚焦显微图像,具体而言:(a)用Cy标记的适配体处理KPL4、HER2阳性乳腺癌细胞系;(b)在N87癌细胞系中使用相同的适配体进行处理;(c)在SK-BR-3癌细胞系中使用相同的适配体进行处理(标记DAPI:蓝色,Cy5-适配体:红色)。
图6示出了用R-[ERBB2适配体]-X-hy(bp)-Cy5分别处理KPL4、N87和SK-BR细胞系的FACS分析结果。
在这方面,表3显示了R-[ERBB2适配体]-ODN-X(R=H、胆固醇或PEG,并且X=H或idT)和cODN-L-F18(L=连接子(linker))的杂交结构,其由R-[ERBB2适配体]-X-hy(bp)-L-F18表示。
图7示出了[AP001-24]-hy(bp)-L-F18的microPET图像的结果。
图8示出了[AP001-24]-idT-hy(bp)-L-F18的microPET图像的结果。
图9示出了胆固醇-[AP001-24]-hy(bp)-L-F18的microPET图像的结果。
图10示出了胆固醇-[AP001-24]-idT-hy(bp)-L-F18的microPET图像的结果。
图11示出了PEG化的-[AP001-24]-hy(bp)-L-F18的microPET图像的结果。
图12示出了PEG化的-[AP001-24]-idT-hy(bp)-L-F18的microPET图像的结果。
图13是在患有KPL4癌症的小鼠中[AP001-24]-hy(bp)-L-F18、[AP001-24]-idT-hy(bp)-L-F18、胆固醇-[AP001-24]-hy(bp)-L-F18和胆固醇-[AP001-24]-idT-hy(bp)-L-F18的比较图片。
图14示出了通过蛋白质印迹确定人乳腺癌细胞系中HER2表达的程度。
图15示出了使用HER2抗体和ERBB2适配体(AP001-25)的乳腺癌细胞系的流式细胞术分析,其中:(a)粘度表显示来自抗体对带有ERBB2适配体(AP001-25)(红色)或对照组DNA序列(蓝色)的BT474(HER2阳性细胞系)和MDA-MB231(HER2阴性细胞系)的荧光信号;(b)包括使用抗体、ERBB2适配体(AP001-25)和阴性对照的两种细胞系的流式细胞术分析图。
图16示出了HER2阳性细胞系中所选ERBB2适配子(AP001-25)的共聚焦显微镜图像,具体而言:(a)用FITC标记适配体处理BT474、HER2阳性乳腺癌细胞系;并且(b)在MDA-MB231癌细胞系中使用相同的ERBB2适配体(AP001-25)处理(标记DAPL:蓝色,FITC-ERBB2适配体:绿色)。
图17示出了代表患有BT474癌症的小鼠中18F-标记的ERBB2适配体{[AP001-25]-hy(bp)-L-F18}的体内PET图像,其中Hy(bp)表示作为杂交的ODN/cODN杂交(碱基配对)。
图18示出了在患有癌症的小鼠中18F-标记的ERBB2适配体{[AP001-25]-hy(bp)-L-F18}的体内分布的研究结果,其中以以百分比计每克组织的活性注射给出数据(%ID/g)(误差线,SD(N=4))。
图19示出了分别在患有HER2阳性和阴性癌症的小鼠中代表18F-标记的ERBB2适配体{[AP001-25]-hy(bp)-L-F18}的体内PET图像,具体而言:(a)HER2过表达的BT474癌症(左腋窝);(b)HER2阴性的MDA-MB231癌症(右腋窝),其中(a)显示比在(b)更多的摄入量;(c)使用18F标记的ERBB2适配体计算进入肿瘤组织的每分钟计数(CPM)和每克的注射量(%ID/g)。
图20示出了HER2的H&E和IHC染色(原始放大400倍)。
具体实施方式
在本发明中使用的与乳腺癌相关的HER2受体特异性结合的ERBB2适配体具有5’-TCAGCCGCCAGCCAGTTC-[核心序列]-GACCAGAGCACCACAGAG-3’的DNA序列,其中核心序列中的数字‘6’或所附DNA序列表中的‘n’代表萘基dU(NaptyldU)。
[表1]
6=NapdU[5-(N-萘羧酰胺)-[0070]2’-脱氧尿苷](NapdU[5-(N-Naphthylcarboxyamide)-[0070]2’-deoxyuridine])。在本发明中,用放射性的同位素(“放射性同位素”)例如18F、32P、123I、89Zr、67Ga、201Tl和111In-111,或荧光染料例如花青荧光染料如Cy3、Cy5、Cy7等标记的HER2适配体用于体内成像。在本发明的实施方案中,已经使用放射性同位素或荧光染料标记的ERBB2适配体对体内分子成像的靶标特异性和潜在的临床应用进行了评估。
用于人表皮生长因子受体2(HER2)的ERBB2适配体用18F-氟化物同位素标记。为了证实适配体进入表达HER2的癌细胞系,通过流式细胞术和共聚焦显微镜将该适配体与对照适配体进行比较。对18F-标记的HER2-特异性ERBB2适配体进行正电子断层扫描,从而获得随时间推移的移植了BT474或KPL4细胞的小鼠的生物分子图像。
【发明的优选实施方案】
在下文中,将详细描述本发明。
细胞培养
表达HER2的人乳腺癌细胞系例如BT474、KPL4、N89和SK-BR-3用于体外和体内实验。此外,将人乳腺癌细胞系MDA-MB231用作对照组。所有细胞系均购自ATCC,并在含有10%FBS的MEM培养基中培养和维持。
细胞裂解,蛋白质印迹
为了提取细胞内蛋白,将包含蛋白酶抑制剂的细胞裂解物在冰上孵育30分钟。通过在4℃下离心20分钟来纯化所得的细胞裂解物。为了进行蛋白质定量,通过Bradford方法对细胞裂解物进行定量,然后使用10%SDS-PAGE通过电泳从各个样品中分离出30μg蛋白质提取物。然后,将所得产物转移至硝酸纤维素膜上,并使用HER2抗体和对照组即β-肌动蛋白抗体作为探针,用ECL在X射线胶片上进行光敏化。
ERBB2适配体合成
靶向HER2-(+)的ERBB2适配体的DNA序列示于下表2中。
[表2]
RBB2适配体、特别是AP001-24与靶标的结合亲和力(Kd)为3.1nM,AP001-25与靶标的结合亲和力为0.9nM。
此处,6表示由下式表示的NapdU[5-(N-萘羧酰胺)-2′-脱氧尿苷],A=2′-脱氧腺苷,G=2′-脱氧鸟苷,并且C=2′-脱氧胞苷。
对于适配体杂交,进行了包括完全匹配序列即在位于每个ERBB2适配体{[AP001-24]和[AP001-25]}的3'的ODN(5'-CAGCCACACCACCAG-3')的合成。
[AP001-24]-ODN合成
如下合成5’-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-3’{[AP001-24]-ODN}。
适配体合成通过亚磷酰胺偶联反应通过固相合成方法进行,并且在合成之后,产物在70℃下在叔丁胺:甲醇:水(1:1:2v/v/v)溶液中反应5小时,从而通过裂解和脱保护过程获得完整的适配体,然后将其干燥。通过HPLC[C18柱(Waters,Xbridge OST C18 10x50mm,260nm]分离合成的适配体,然后通过ESI MS质谱仪(Qtrap2000,ABI)进行分子量测量。
表1中的第11个适配体(SEQ ID NO:11)对应于AP001-24。
[AP001-25]-ODN合成:
通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的合成步骤合成5’-[A6G 66A GAG 666GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-3’{[AP001-25]-ODN}。
表1中的第12个适配体(SEQ ID NO:7)对应于AP001-25。
以相同的方式,通过与用于{[AP001-24]-ODN}合成的章节中所述的相同步骤合成每种适配体,即CAG-3'{表1-ODN中的每个适配体(SEQ ID NO:1-35)}。
[AP001-24]-ODN-idT合成:
使用idT(反向dT)CPG(Glen,20-0302-10)通过与用于{[AP001-24]-ODN}合成的章节中所述的相同步骤合成5’-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAGA]-CAG CCA CAC CAC CAG-idT-3’{[AP001-24]-ODN-idT}。
[AP001-25]-ODN-idT合成:
使用idT CPG(Glen,20-0302-10)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACAA]-CAG CCA CAC CAC CAG-idT-3’{[AP001-25]-ODN-idT}。
胆固醇基-[AP001-24]-ODN合成:
使用胆固醇-PA(Glen,10-1976-90)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-胆固醇基-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66ACA GCC CAG A]-CAG CCA CAC CAC CAG-3’{胆固醇基-[AP001-24]-ODN}。
胆固醇基-[AP001-25]-ODN合成:
使用胆固醇-PA(Glen,10-1976-90)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-胆固醇基-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAGGGC G6A ACA A]-CAG CCA CAC CAC CAG-3’{胆固醇基-[AP001-25]-ODN}。
胆固醇基-[AP001-24]-ODN-idT合成:
使用idT CPG(Glen,20-0302-10)和胆固醇基-PA(Glen,10-1976-90)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-胆固醇基-[6CC 6GG CA6G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-idT-3’{胆固醇基-[AP001-24]-ODN-idT}。
胆固醇基-[AP001-25]-ODN-idT合成:
使用idT CPG(Glen,20-0302-10)和胆固醇-PA(Glen,10-1976-90)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-胆固醇基[A6G 66A GAG 666GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-idT-3’{胆固醇基-[AP001-25]-ODN-idT}。
PEG化的-[AP001-24]-ODN合成:
使用聚乙二醇2000 CED PA(ChemGenes,CLP-2119)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-PEG化的-[6CC 6GG CA6 G66 CGA 6GG AGGCC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG’{PEG化的-[AP001-24]-ODN}。
PEG化的-[AP001-25]-ODN合成:
使用聚乙二醇2000 CED PA(ChemGenes,CLP-2119)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-PEG化的-[A6G 66A GAG 666 GCC 6GA G6GCC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-3’{PEG化的-[AP001-25]-ODN}。
PEG化的-[AP001-24]-ODN-idT合成:
使用idT CPG(Glen,20-0302-10)和聚乙二醇2000 CED PA(ChemGenes,CLP-2119)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-PEG化的-[6CC6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-idT-3’{PEG化的-[AP001-24]-ODN-idT}。
PEG化的-[AP001-25]-ODN-idT合成:
使用idT CPG(Glen,20-0302-10)和聚乙二醇2000 CED PA (ChemGenes,CLP-2119)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-PEG化的-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CACCAG-3’{PEG化的-[AP001-25]-ODN-idT}。
缀合Cy5的cODN(互补寡核苷酸)[cODN-Cy5]合成:
下图代表cODN-Cy5和cODN-L-F18(L=连接子)的结构及其合成。
使用Cy5-PA(Glen,10-5915-10)通过与上述用于{[AP001-24]-ODN}合成的章节中所述的相同的步骤合成5’-Cy5-[CTGGTGGTGTGGCTG]-3’[cODN-Cy5]。
形成cy5-标记的ERBB2适配体
表3显示了R-[ERBB2适配体]-ODN-X(R=H、胆固醇或PEG,并且X=H或idT)和cODN-Cy5的杂交结构,其由[ERBB2适配体]-X-hy(bp)-Cy5表示。
[表3]
以下面的方式制备Cy5-标记的ERBB2适配体即{R-[ERBB2适配体]-X-hy(bp)-Cy5}。
首先,将等摩尔的cODN-Cy5和[ERBB2适配体]-ODN放入退火缓冲液(PBS)中。在此,将MgCl2的浓度控制为达到10mM的最终浓度。将该反应产物在95℃下放置5分钟,然后在室温下缓慢冷却。cODN-Cy5和[ERBB2适配体]-ODN的杂交效率通过电泳(Typhoon FLA 70003%琼脂糖凝胶分析)和HPLC(XBridge OST分析柱(2.5μm,4.6×50mm,Waters,254nm,0.1MTEAA/乙腈)进行评价。图2显示了使用Typhoon FLA 7000 3%琼脂糖凝胶分析R-[ERBB2适配体]-X-hy(bp)-Cy5的结果,R-[ERBB2适配体]-X-hy(bp)-Cy5是通过[ERBB2适配体]-ODN-X(R=H、胆固醇或PEG,并且X=OH或idT)与cODN-Cy5杂交的产物。
评估了用荧光染料即Cy5标记的合成寡核苷酸(cODN-Cy5)与[ERBB2适配体]-ODN之间的互补碱基配对。将胆固醇-[AP001-24]-ODN-idT或胆固醇-[AP001-24]-ODN和cODN-Cy5以1:1比例混合后,保持温度,使这些成分在55、60和65℃结合在一起。为了确认结合,在3%琼脂糖凝胶中进行电泳,然后通过FLA 5000对Cy5进行荧光成像。然后,将整个适配体用EtBr染色,并进行UV成像。结果显示在图3中。为了比较胆固醇基-[AP001-24]-ODN-idT、胆固醇基-[AP001-24]-ODN、PEG化[AP001-25]-ODN-idT或PEG化[AP001-25]-ODN和cODN-Cy5之间的互补碱基配对,将这些适配体中的每一个与cODN-Cy5以1:1的比例混合,并在95℃加热5分钟以结合在一起,然后以与上述相同的方式评估结合。在有或没有在95℃下加热的情况下测定胆固醇基-[AP001-24]-ODN-idT、胆固醇基-[AP001-24]-ODN、PEG化-[AP001-25]-ODN-idT或PEG化-[AP001-25]-ODN与cODN-Cy5之间的互补碱基配对,并将其相互比较。比较结果显示在图4中。
形成F18放射性同位素-标记cODN(互补寡核苷酸)[cODN-L-F18]
基于现有技术中早已报道的方法进行18F-标记的cODN的合成(参见参考文献24)。在合成装置(Tracerlab FXFN,GE Healthcare,Milwaukee,WI,USA)中生成不添加载体的18F氟离子后,将其与甲磺酸酯反应(在100℃持续10分钟),然后通过使用HPLC纯化18F-氟-PEG-叠氮化物(18F-FPA)。将1M N,N-二异丙基乙胺的乙腈溶液(10mL)和100mM碘化铜的乙腈溶液(20mL)加入5'-己炔基互补寡核苷酸(5'-hex-cODN;200mg),向其中进一步添加18F-FPA(750e1100MBq),然后进行点击化学反应(在70℃下20分钟)。合成的18F-标记的cODN(cODN-L-F18)使用HPLC H(Xbridge OST C18 10x 50mm,洗脱液为在20分钟5:95至95:5的乙腈/0.1M TEAA,流速5mL/min,并且UV(254nm))纯化。
形成F18放射性同位素-标记的ERBB2适配体{R-[ERBB2适配体]-X-hy(bp)-L-F18]
下表4显示了R-[ERBB2适配体]-ODN-X(R=H、胆固醇或PEG,并且X=OH或idT)和cODN-L-F18(L=连接子)的杂交结构,其由[ERBB2适配体]-X-hy(bp)-L-F18表示。
[表4]
以下面的方式制备F18放射性同位素-标记的ERBB2适配体{R-[ERBB2适配体]-X-hy(bp)-L-F18}。
首先,将等摩尔的cODN-L-F18和[ERBB2适配体]-ODN放入退火缓冲液(PBS)中。在此,将MgCl2的浓度控制为达到10mM的最终浓度。将该反应产物在95℃下放置5分钟,然后在室温下缓慢冷却。cODN-L-F18和[ERBB2适配体]-ODN的杂交效率使用HPLC(XBridge OST分析柱(2.5μm,4.6x 50mm,Waters,254nm,0.1M TEAA/乙腈)进行评价。这些产物以98%或更高的杂交率合并。
共聚焦显微镜
将BT474、KPL4、N87、SK-BR-3和MDA-MB231细胞系分配在盖玻片上并孵育过夜。当约80%的细胞系生长时,小心洗涤生长的细胞,并通过使用浓度为250mM的荧光标记的ERBB2适配体{R-[ERBB2适配体]-hy(bp)-Cy5}进行处理来孵育。培养后,将产物仔细洗涤,然后将载有DAPI的培养基装载在载玻片上。然后,通过LSM 700共聚焦显微镜观察其荧光。显微镜设置如下:用488激光进行FITC观察;使用BP490-555观察到激发和发射;639激光用于德克萨斯红;并且使用LP640滤光片观察到发射。
以与先前实验相同的方式,将过表达ERBB2的乳腺癌细胞系例如KPL4、N87和SK-BR-3分配在盖玻片上并孵育过夜。当约80%的细胞系生长时,小心洗涤生长的细胞,并使用互补碱基配对,使用结合至ERBB2适配体的Cy5荧光标记的ODN制备的样品通过处理进行孵育。培养后,将产物仔细洗涤,然后将载有DAPI的培养基装载在载玻片上。然后,通过LSM700共聚焦显微镜观察荧光。
观测结果显示在图5中。
流式细胞术
ERBB2适配体的特异性通过使用流式细胞仪系统(BD Biosciences)的荧光激活细胞分离方法来验证。在陪替氏培养皿上继代培养适当数量的BT474、KPL4、N87、SK-BR-3或MDA-MB231癌细胞系,使其生长到大约80%。用胰蛋白酶处理生长的细胞,并用PBS洗涤,然后通过根据温度产生的互补碱基将荧光标记的ODN与ERBB2适配体结合。用结合完成的样品处理细胞。将ERBB2适配体{R-[ERBB2适配体]-hy(bp)-Cy5}和对照组即含1%FES的抗体,分别在4℃下处理30分钟。洗涤完全处理的样品,然后通过荧光活化细胞分离法测量和分析结合的ERBB2适配体。
上述测量和分析的结果示于图6中。
体内实验
将17ββ-雌二醇沉淀物皮下植入一只四周大的Balb/c裸鼠的颈部侧面区域,以便释放出足够量的雌激素,以潜在地诱发癌症。几天后,将BT474或KPL4人乳腺癌细胞系皮下植入每只小鼠7x 106个细胞中。在癌症发展3周后,使用卡尺测量癌症的生长。
在Balb/C裸鼠的右肩中,将作为人类乳腺癌细胞系的KPL4细胞皮下植入每只小鼠1x 105个细胞中。此后,诱发癌症的发生。
F18放射性同位素-标记的ERBB2适配体的PET成像
将F18放射性同位素-标记的ERBB2适配体注入小鼠体内,60分钟后,通过InveronmicroPET扫描仪(Siemens,Knoxville,TN,USA)扫描10分钟获得静态图像。对于F18放射性同位素-标记的ERBB2适配体注射,小鼠用2%异氟烷麻醉,然后将7.4MBq的F18放射性同位素-标记的ERBB2适配体注射入尾静脉。将获得的列表模式数据转换为正弦图,并通过3D有序子集期望最大化(Ordered Subset Expectation Maximization,OSEM)算法进行重新配置,然后使用ASIpro(Concord Microsystems Inc.,Knoxville,TN)进行评估。
将F18放射性同位素-标记的ERBB2适配体静脉内注射给患有通过注射人乳腺肿瘤细胞而生长的肿瘤的小鼠后,使用西门子的Inveon PET(Knoxville,TN)进行PET。注入量为13.7±1.1MBq(370±30uCi),并根据十个1分钟图像和四个5分钟图像方案进行了动态PET研究30分钟。注射后分别进行了10、60、90和120分钟的这两个静态研究。由AMIDE软件执行PET信号的部分定量。图像实际上是通过与正电子标记探针的组织浓度(%ID/g)成比例的假色标度获得的。红色代表最高浓度,而黄色、绿色和蓝色对应逐渐降低的浓度。
PET图像显示在图7至13中。
结果
验证HER2表达和适配体对靶肿瘤细胞的亲和力
进行了蛋白质印迹和流式细胞术以研究HER2在乳腺癌细胞系BT474中的表达。通过蛋白质印迹分析,证实了由于基因扩增而导致已知过度表达HER2的BT474以及SKBR3细胞系的过度表达。此外,发现在阴性对照细胞系MDA-MB231(图14)的相应部位未检测到信号。
如图15所示,使用流式细胞术证明HER2抗体与HER2阳性BT474细胞系非常特异性结合。与抗体相比,可以看出ERBB2适配体在MDA-MB231细胞系中非常弱,而在BT474细胞系中它与之牢固结合。此外,在随机寡核苷酸中看不到适配体与任何细胞系的结合。这些结果表明,ERBB2适配体优先结合于HER2阳性细胞系,并且这种结合可以通过识别细胞系表面上的HER2结构来实现。以相同的方式,观察到作为乳腺癌细胞系的KPL4与SK-BR-3细胞系荧光标记的适配体牢固结合。
共聚焦显微镜分析
通过共聚焦显微镜进一步评估ERBB2适配体与细胞的结合(图16)。用适配体处理BT474 HER2阳性乳腺癌细胞系。由于ERBB2适配体被荧光标记,因此在细胞表面观察到荧光,并且鉴定了存在于细胞表面的HER2结构。沿细胞膜观察到适配体显示的荧光,而作为阴性对照组的MDA-MB231细胞系未显示任何荧光信号,因此被确定为不包括HER2。因此,观察到ERBB2适配体可以与HER2阳性乳腺癌细胞系结合,但是与HER2阴性细胞结合最少。用ERBB2适配体{[AP001-24]和[AP001-25]}处理乳腺癌细胞系KPL4、N87和SK-BR-3后,它们与荧光标记的ODN形成互补碱基配对,然后使用共焦显微镜通过与上述实验相同方法进行荧光观察。发现上述两种类型的ERBB2适配体均与乳腺癌细胞系结合良好,其中[AP001-24]在沿细胞膜的细胞表面上显示荧光,而[AP001-25]甚至在细胞内也显示荧光。
体内PET成像、体内分布、免疫组织化学
根据动物微型PET,随时间给出了患有BT474或KPL4癌症的小鼠的体内生物分子图像。参见图17,观察到在小鼠左腋窝中存在的肿瘤组织中18F-标记的HER2特异性ERBB2适配体的摄入显著增加。在120分钟的图像中,ERBB2适配体在水平图像和冠状图像中清楚地标记了癌症。肠道和膀胱中明显出现的生理摄入可能反映了这些器官是放射医学的两种主要释放途径。
注射18F-标记的ERBB2适配体后1小时,在患有癌症的小鼠中验证了体内分布。处死动物后,通过γ计数器测量包括癌症在内的单独组织中的辐射水平,然后以%ID/g表示(图18)。测量结果还显示在下表5中。
[表5]
癌症中18F-标记的ERBB2适配体的摄入量为每小时0.62±0.04。体内分布的研究表明,肾和肠道是18F-标记的ERBB2适配体的两条主要释放途径。
图19示出分别在患有HER2阳性和阴性癌症的小鼠中的18F-标记的ERBb2适配体的图像。相比之下,过表达HER2的BT474癌症的同位素摄入量高于HER2阴性的MSA-MB231癌症。出于半量化的目的,计算了VOI(感兴趣的体素或体积)中的总活性(nCi)。作为比较BT474和MDA-MB231细胞系之间的T/M(肿瘤/肌肉)摄取比率的结果,在BT474癌症中过表达的HER2显示出更高的T/M比率和对比图像(图19)。在免疫组织化学方面,已经证实,从单独的小鼠组切除的BT474癌症显示出高表达HER2,而MDA-MB231细胞具有较低的HER2表达(图20)。相比之下,观察到BT474癌细胞(上排)显示出比MDA-MB231细胞系(下排)更高的细胞膜中HER2染色。
根据本发明,靶向HER2的ERBB2适配体已成功地在体内PET成像。本发明是首例使用ERBB2特异性适配体进行HER2靶PET成像。在患有BT474癌症的小鼠中,PET图像显示ERBB2适配体可能在体内识别HER2,并且相对有特色地显示出该癌症。基于这些结果,放射性标记的ERBB2适配体可用于HER2阳性乳腺癌细胞系的靶向治疗,或潜在地用于确定其合适的治疗方法。
如以上实施方案中所鉴定的,当通过将R-[ERBB2适配体]-ODN-X与cODN-L-F18结合制备R-[ERBB2适配体]-ODN-X/cODN-L-F18(在以上描述中由R-[ERBB2适配体]-X-hy(bp)-L-F18表示)时,例如从R=H(无保护基)和X=H(无保护基)到R=胆固醇或PEG(聚乙二醇)和X=idT(倒置脱氧胸苷)、LNA(锁核酸)、2'-甲氧基核苷酸、2'-氨基核苷酸、2'F-核苷酸等例如在5'末端或3'末端位置或这两个末端位置化学修饰(即受保护)的适配体可以确保获得更好的图像。
因为由于上述化合物的修饰可以改善增加t1/2(半衰期)血液清除率的效果,即增加在血液中的体内半衰期,因此具有放射性同位素结合的ERBB2适配体越来越多地与肿瘤结合,因此提高成像效率[与当R=H和X=H时的t1/2=10分钟相比,如果保护和修饰R和X,t1/2则增加到1小时,从而显示出更好的图像]。
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<213> 人工序列
<220>
<223> 核心序列由克隆编号9-ER-N-F11_G09表示,其中n是NapdU [5- (N-萘羧酰胺)-2’-脱氧尿苷]
<400> 31
ngagaagggcngngccnnacncaaaannnggggancngaa 40
<210> 32
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 核心序列由克隆编号9-ER-N-G04_B10表示,其中n是NapdU [5- (N-萘羧酰胺)-2’-脱氧尿苷]
<400> 32
cgnccnnggngagnnngggncngagcaggagcacgngagn 40
<210> 33
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 核心序列由克隆编号9-ER-N-H01_E10表示,其中n是NapdU [5- (N-萘羧酰胺)-2’-脱氧尿苷]
<400> 33
annagangaaagcacannccaacaacaganaancngaggg 40
<210> 34
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 核心序列由克隆编号9-ER-N-H03_G10表示,其中n是NapdU [5- (N-萘羧酰胺)-2’-脱氧尿苷]
<400> 34
annagangaaagcacannccaacaacaganaancngaggg 40
<210> 35
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 核心序列由克隆编号9-ER-N-H09_B11表示,其中n是NapdU [5- (N-萘羧酰胺)-2’-脱氧尿苷]
<400> 35
angnnagagncngccngagngccncgcaagggcgnaacag 40
17
Claims (10)
1.一种用于对肿瘤疾病区域成像的组合物,其包括HER2特异性ERBB2适配体,所述适配体包含:选自序列表编号(SEQ ID NO)1至35的DNA序列,其中所述适配体用放射性同位素或荧光染料标记。
2.根据权利要求1所述的组合物,其中,所述适配体包括具有以下SEQ ID NO:AP001-24或SEQ ID NO:AP001-25的DNA序列:
AP001-24:5’-A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A-3’,和
AP001-25:5’-6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A-3’,其中6是NapdU[5-(N-萘羧酰胺)-2’-脱氧尿苷],A=2’-脱氧腺苷,G=2’-脱氧鸟苷,并且C=2’-脱氧胞苷。
3.根据权利要求1所述的组合物,其中,所述适配体是在5′-末端位置和3′-末端位置中的至少一个位置上化学修饰的适配体。
4.根据权利要求3所述的组合物,其中,通过胆固醇或PEG(聚乙二醇)进行所述修饰。
5.根据权利要求3所述的组合物,其中,通过反向脱氧胸苷(idT)、锁核酸(LNA)、2'-甲氧基核苷酸、2'-氨基核苷酸或2'F-核苷酸进行所述修饰。
6.根据权利要求1所述的组合物,其中,所述放射性同位素选自18F、32P、123I、89Zr、67Ga、201Tl和111In-111。
7.根据权利要求6所述的组合物,其中,所述放射性同位素是18F。
8.根据权利要求1所述的组合物,其中,所述荧光染料是花青荧光染料。
9.根据权利要求8所述的组合物,其中,所述荧光染料是Cy5。
10.一种提供癌症诊断信息或癌症转移诊断信息的方法,包括:
将患者的生物样品与根据权利要求1至9中任一项所述的标记的适配体反应;
测量所述适配体在所述患者的所述生物样品中的结合程度;和
比较所述适配体在所述患者的所述生物样品中的结合程度与所述适配体在其正常样品中的结合程度。
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