CN115337413B - 一种针对非小细胞肺癌诊断的放射性分子探针的制备及应用 - Google Patents
一种针对非小细胞肺癌诊断的放射性分子探针的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种针对非小细胞肺癌的特异靶向性放射性分子探针的制备及应用。包括用于结合ANGPT2靶点的单克隆抗体、螯合剂以及放射性核素;所述放射性分子探针具体可为99mTcO‑DTPA‑Nesvacumab。本发明所制备的分子探针作为显像药物,肿瘤靶向性强,显像明显,易于观察;本发明中使用DTPA作为螯合剂,体内和体外的稳定性强,便于应用;本发明提高了非小细胞肺癌的诊断效果,为非小细胞肺癌的高效诊疗一体化提供了新思路。
Description
技术领域
本发明属于生物医药领域,具体涉及一种针对非小细胞肺癌的特异靶向性放射性分子探针的制备及应用。
背景技术
肺癌作为我国最常见的癌症之一,其生存率不到30%,且病人以中晚期患者居多,早期病人较少,主要原因在于发现时间晚,延误治疗效果,癌症如可以早期发现,治愈率可达80%以上。放射性示踪剂作为广泛使用的肿瘤靶向显像工具,可以提供疾病的功能信息并跟踪体内的生化过程。同时,作为诊断和治疗癌症的三大手段之一,用于诊断的放射性药物通常具有较高的检测灵敏度,仅需很小的剂量即可通过PET或SPECT显像技术对患者进行无创精准的诊断。因此,开发对肿瘤具有靶向能力和高灵敏度的分子探针可以在一定程度上对癌症进行诊断并进行更准确的治疗。
新生血管形成过程是癌症成长所必须的要素,抑制血管生成是抑制癌症的重要策略,血管生成素2(Angiopoietin 2,ANGPT2)是近年来发现一种分泌型的生长因子,具有促血管生成的作用,通过调节血管的生长和抑制使其处于平衡状态。ANGPT2在非小细胞肺癌组织中高表达,具有发展为非小细胞肺癌诊断和治疗标志物的潜力。
发明内容
为了解决非小细胞肺癌显像和诊断的实际问题,本发明设计并合成了一种对非小细胞肺癌具有特定靶向作用的放射性分子探针,该分子探针将针对ANGPT2的单抗通过偶联键与螯合剂进行偶联,在螯合剂上标记放射性核素,利用SPECT/CT实现对非小细胞肺癌的精准诊断,在肺癌的高效诊断和治疗研究中显示出潜在的应用前景。
为了实现上述发明目的,本发明提供一种针对ANGPT2的放射性分子探针。
本发明所提供的针对ANGPT2的放射性分子探针,包括用于结合ANGPT2靶点的单克隆抗体、螯合剂以及放射性核素,其中,单克隆抗体与螯合剂共价偶联,放射性核素缀合到螯合剂上。
所述用于结合ANGPT2靶点的单克隆抗体具体可为(Nesvacumab,奈伐苏单抗,卡梅德(天津)生物科技,货号YR1297);
所述螯合剂包括但不限于以下:
所述放射性核素选自99mTcO、18F、89Zr、68Ga中的任意一种,优选99mTcO、68Ga或18F;
所述螯合剂为DTPA,所述放射性核素为99mTcO,所述单抗为Nesvacumab,所述放射性分子探针表示为99mTcO-DTPA-Nesvacumab。
上述99mTcO-DTPA-Nesvacumab放射性分子探针通过包括如下步骤的方法制备得到:
1)将DTPA活化;
2)将活化后的DTPA与Nesvacumab偶联,得到抗体标记物;
3)将所得抗体标记物溶于缓冲溶液中,加入氯化亚锡溶液,加入99mTcO生理盐水溶液,反应,得到放射性分子探针99mTcO-DTPA-Nesvacumab。
上述方法步骤1)中,采用碳二亚胺为活化剂将DTPA活化,
其中,DTPA与活化剂的摩尔比可为1:2-20,活化时间可为1-4h,活化温度可为10℃-60℃;
在本发明的一个实施方案中,DTPA与活化剂的摩尔比为1:10,室温活化2h;
上述方法步骤2)中,DTPA与Nesvacumab的摩尔比可为:8-12:1,具体可为10:1;
所述偶联在缓冲溶液中进行;
所述缓冲溶液为pH为8.0-10.0的碳酸盐缓冲液;
所述偶联的温度可为10℃-60℃,时间可为10min-60min;
在本发明的一个实施方案中,所述偶联在pH为8.5的碳酸盐缓冲液中在室温下偶联30min,
偶联反应完成后,可进一步包括将所得体系在PBS缓冲液中透析得到产物的操作,
其中,透析用PBS缓冲液的浓度可为0.01M-0.5M;
在本发明的一个实施方案中,所述透析用PBS缓冲液的浓度可为0.01M。
上述方法步骤3)中,所述缓冲溶液为0.1M PBS缓冲液或生理盐水,pH为5至8,
所述氯化亚锡溶液的浓度可为1mg/mL,加入1mg/mL的氯化亚锡(DTPA-Nesvacumab产物与氯化亚锡的摩尔比为5:1-1:2,具体可为1:1),加入99mTcO生理盐水溶液(50-200μCi,具体可为100μCi)后反应时间可为10min-60min,反应温度可为10—60℃,
在本发明的一个实施方案中,所述缓冲溶液为0.1M PBS缓冲溶液,pH为6.5;
在本发明的一个实施方案中,加入1mg/mL的氯化亚锡,加入99mTcO生理盐水溶液后室温反应15min得到放射性分子探针99mTcO-DTPA-Nesvacumab。
上述针对ANGPT2的放射性分子探针(具体可为99mTcO-DTPA-Nesvacumab)在制备非小细胞肺癌诊断试剂中的应用也属于本发明的保护范围。
本发明具有如下有益效果:
本发明所述的放射性分子探针(具体可为99mTcO-DTPA-Nesvacumab)与现有技术相比较的有益效果包括:
1)本发明通过简单快速的方法制备该放射性分子探针,合成方法简便快捷,有利于实际应用;
2)本发明所制备的分子探针作为显像药物,肿瘤靶向性强,显像明显,易于观察;
3)本发明中使用DTPA作为螯合剂,体内和体外的稳定性强,便于应用;
4)本发明提高了非小细胞肺癌的诊断效果,为非小细胞肺癌的高效诊疗一体化提供了新思路。
本发明针对ANGPT2特异性结合单抗,设计了一种新型靶向非小细胞肺癌的放射性分子探针,可用于非小细胞肺癌的显像和指导治疗。
附图说明
图1为流式细胞仪分析A549细胞对Nesvacumab的摄取效果,横坐标表示荧光强度。
图2为FITC-Nesvacumab经裸鼠尾静脉注射后,荧光活体成像观察A549细胞皮下移植瘤以及正常器官对Nesvacumab的摄取情况。*p﹤0.05,#p﹤0.001。
图3为FITC-Nesvacumab经裸鼠尾静脉注射后,正常器官及A549细胞皮下移植瘤摄取Nesvacumab的荧光强度分析。*p﹤0.05,#p﹤0.001。
图4为FITC-Nesvacumab经裸鼠尾静脉注射后,组织学观察不同时间点A549细胞皮下移植瘤对Nesvacumab的摄取情况。
图5为DTPA-Nesvacumab放射性分子探针高效液相色谱(HPLC)图谱。
图6为99mTcO-DTPA-Nesvacumab放射性分子探针高效液相色谱(HPLC)图谱。
图7为BABL/c裸鼠模型空白小鼠尾静脉注射99mTcO生理盐水溶液的SPECT显像结果,图中从左至右依次为注射后0.5、1、1.5、2和3h的显像结果。
图8为BABL/c裸鼠模型肺癌小鼠注射99mTcO-DTPA-Nesvacumab的SPECT显像结果,图中从左至右依次为注射后0.5、1、1.5、2和3h的显像结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中采用的试剂为:
实施例1、A549细胞对Nesvacumab的摄取分析
1)荧光化合物FITC-Nesvacumab的合成
将Nesvacumab在0.01M碳酸盐缓冲液(PH=8.5)体系透析,按照1:10摩尔比例将FITC(异硫氰酸荧光素)加入到上述透析产物中,室温反应30min后,用0.01MPBS透析过夜,即得抗体的FITC标记物。
2)细胞摄取分析
人脐静脉内皮细胞HUVEC细胞和A549按照1:5的比例分别铺于Transwell的上、下室,培养48小时模拟肿瘤微血管与肿瘤细胞环境,将Transwell上室中的HUVEC细胞培养液吸出,加入含10%FBS的培养基(FITC-Nesvacumab的终浓度为0.5μg/ml),37℃孵育,分别于4和8h用0.25%胰蛋白酶消化并用冷PBS洗涤3次,然后重悬于300μl冷PBS中,用于流式测定细胞荧光强度。结果显示8h时A549细胞的FITC荧光强度大于4h和PBS组,说明Nesvacumab能够穿过HUVEC细胞被A549摄取(图1)。
实施例2、荷A549细胞皮下移植瘤裸鼠对Nesvacumab的摄取分析
1)空白BABL/c荷瘤裸鼠脏器和瘤体对Nesvacumab的摄取
每只正常荷瘤裸鼠,尾静脉注射实施例1的FITC-Nesvacumab溶液100μl(0.7μg/μl)与等体积的PBS(100μl),注射后12h分离小鼠脏器,使用小动物荧光活体成像仪采集图像(图2),结果显示,注射PBS的空白荷瘤裸鼠的脏器不显示FITC荧光。
2)A549细胞皮下移植瘤BABL/c裸鼠脏器和瘤体对Nesvacumab的摄取
右前肢皮下接种非小细胞肺癌细胞A549细胞的荷瘤鼠,尾静脉注射实施例1的FITC-Nesvacumab溶液100μl(0.7μg/μl),注射后3、12、24、48h分别分离小鼠脏器和瘤体,使用小动物活体成像仪采集图像,结果显示,单独注射实施例2的探针时,在3h,相对于脏器,肿瘤组织对Nesvacumab的摄取最高(图2、图3),说明Nesvacumab对A549细胞移植瘤的最佳显像时间为3h内。
3)A549细胞皮下移植瘤摄取Nesvacumab的组织学观察
空白荷瘤和不同时间点的ANGPT2单抗摄取荷瘤经4%多聚甲醛固定、梯度酒精脱水和浸蜡,后制备成石蜡包埋组织。利用组织切片机切出厚度为4μm的组织切片,经脱蜡、梯度酒精脱水,用5μg/mL的4',6-二脒基-2-苯基吲哚(4’,6-diamidino2-phenylindole,DAPI)染色细胞核,最后用防淬灭剂封片,在荧光显微镜下拍照观察(图4)。(DAPI指示细胞核、FITC指示Nesvacumab、组合为DAPI与Nesvacumab荧光叠加)由图4可知:从12h开始,肿瘤组织的荧光信号减弱,表明ANGPT2单抗逐步被代谢和清除(图4)。
实施例3、化合物DTPA-Nesvacumab的合成
1)称取适量EDC试剂,用0.01M MES溶解;DTPA与EDC摩尔比为1:10,室温活化2h,将活化的DTPA加入到经过0.01M碳酸盐缓冲液(PH=8.5)透析过的抗体蛋白中(DTPA与Nesvacumab的摩尔比为:10:1),室温反应30min后,用0.01M PBS透析过夜,即得抗体的DTPA标记物。
2)质量控制
使用HPLC对合成的DTPA-Nesvacumab进行分析,包括以下步骤:
C18分析柱(4.6mm×250mm),HPLC梯度洗脱条件:0min,乙腈/水(33/67,v/v);10min,乙腈/水(100/0,v/v);洗脱剂中含有0.1%TFA,流速为1mL/min。从HPLC图谱中观察,DTPA-Nesvacumab的保留时间为2.5min,化学纯度>99%,如图5所示。
实施例4、99mTcO-DTPA-Nesvacumab放射性分子探针的合成
99mTcO-DTPA-Nesvacumab放射性分子探针包括DTPA-Nesvacumab和放射性核素99mTcO,所述Nesvacumab和99mTcO用DTPA连接,其制备方法包括以下步骤:
1)99mTcO的淋洗
用5mL注射器抽取生理盐水5mL,对钼锝发生器进行淋洗,收集淋洗液至离心管内,使用医用活度计对放射性进行检测,活度为5mCi。
2)99mTcO标记DTPA-Nesvacumab
将0.2mL 1mg/mL的氯化亚锡加入至0.2mL99mTcO(100μCi)生理盐水溶液中,混合均匀,加入pH为6.5,实施例3中制得的DTPA-Nesvacumab的PBS缓冲溶液(1mg/mL的DTPA-Nesvacumab,50μL),常温下混合震荡15min。
3)质量控制
使用HPLC对合成的放射性分子探针进行分析,分析条件包括以下步骤:
C18分析柱(4.6mm×250mm),HPLC梯度洗脱条件:0min,乙腈/水(33/67,v/v);34.6min,乙腈/水(100/0,v/v);42min,乙腈/水(33/67,v/v);洗脱剂中含有0.1%TFA,流速为1mL/min。从放射性HPLC图谱中观察,99mTcO-DTPA-Nesvacumab的保留时间为33.4min,放化纯度>99%,如图6所示,原2.5min处DTPA-Nesvacumab的峰基本消失。
实施例5、裸鼠皮下移植瘤显像分析
1)空白BABL/c荷瘤裸鼠的SPECT显像
空白荷瘤鼠,尾静脉注射实施例5的99mTcO生理盐水注射液100μCi,注射后0.5、1、1.5、2和3h分别使用SPECT采集10min静态图像(图7),结果显示,空白荷瘤鼠在注射入放射性核素时,核素主要集中在甲状腺和膀胱两个部位,整个小鼠轮廓明显且均匀,说明核素在小鼠体内分布均匀。
2)A549细胞皮下移植瘤BABL/c裸鼠的SPECT显像
右前肢皮下接种非小细胞肺癌细胞A549细胞的荷瘤鼠,尾静脉注射实施例4制得的99mTcO-DTPA-Nesvacumab注射液100μCi,注射后0.5、1、1.5、2和3h分别使用SPECT采集10min静态图像(图8,其中箭头指示为肿瘤部位),结果显示,在各个时间点,与对侧的正常组织对比,肿瘤部位有明显的放射性摄取,肿瘤清晰可见。在肾脏和膀胱中观察到99mTcO-DTPA-Nesvacumab的显著摄取,表明该示踪剂主要通过泌尿途径排泄。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。
Claims (5)
1.放射性分子探针,包括用于结合ANGPT2靶点的单克隆抗体Nesvacumab、螯合剂以及放射性核素,其中,单克隆抗体Nesvacumab与螯合剂共价偶联,放射性核素缀合到螯合剂上;
所述螯合剂为DTPA,所述放射性核素为99mTcO,所述放射性分子探针表示为99mTcO-DTPA- Nesvacumab;
制备所述的放射性分子探针的方法,包括如下步骤:
1)将DTPA活化;
2)将活化后的DTPA与Nesvacumab偶联,得到抗体标记物;
3)将所得抗体标记物溶于缓冲溶液中,加入氯化亚锡溶液,加入99mTcO生理盐水溶液,反应,得到放射性分子探针99mTcO-DTPA-Nesvacumab。
2.根据权利要求1所述的放射性分子探针,其特征在于:步骤1)中,采用碳二亚胺为活化剂将DTPA活化,
其中,DTPA与活化剂的摩尔比为1:2-20,活化时间为1-4 h,活化温度为10 ℃-60 ℃。
3.根据权利要求1所述的放射性分子探针,其特征在于:步骤2)中,DTPA与Nesvacumab的摩尔比为:8-12:1;
所述偶联在缓冲溶液中进行;
所述缓冲溶液为pH为8.0-10.0的碳酸盐缓冲液;
所述偶联的温度为10 ℃-60 ℃,时间为10 min-60 min。
4. 根据权利要求1所述的放射性分子探针,其特征在于:步骤3)中,所述缓冲溶液为0.1 M PBS缓冲液或生理盐水,pH为5至8,
所述氯化亚锡溶液的浓度为0.5 mg/mL-5 mg/mL,加入99mTcO生理盐水溶液后反应时间为10 min-60 min,反应温度为10 ℃-60 ℃。
5.权利要求1-4中任一项所述的放射性分子探针在制备非小细胞肺癌诊断试剂中的应用。
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