靶向表皮生长因子受体单克隆抗体预定位免疫PET显像的实验研究  

Pretargeted immunoPET imaging with epidermal growth factor receptor monoclonal antibody in mouse models

作　　者：苑陆杰 李慧玲 盖永康 张永学[1] 兰晓莉[1] Yuan Lujie;Li Huiling;Gai Yongkang;Zhang Yongxue;Lan Xiaoli(Department of Nuclear Medicine,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology Hubei Key Laboratory of Molecular Imaging,Wuhan 430022,China)

机构地区：[1]华中科技大学同济医学院附属协和医院核医学科、湖北省分子影像重点实验室,武汉430022

出　　处：《中华核医学与分子影像杂志》2022年第2期74-79,共6页Chinese Journal of Nuclear Medicine and Molecular Imaging

基　　金：国家自然科学基金(81630049)。

摘　　要：目的:采用预定位技术在表皮生长因子受体(EGFR)阳性/阴性荷瘤鼠中探索西妥昔单克隆抗体(简称单抗;Cetuximab)靶向EGFR免疫PET显像的可行性。方法:以反式环辛烯(TCO)-N-羟基琥珀酰亚胺(NHS)修饰Cetuximab获得Cetuximab-TCO。以2,2′-((6-氨基-1-(4,7-双(羧甲基)-1,4,7-三氮烷-1-基)己烷-2-基)氮杂二酰基)二乙酸(L-NETA)为螯合剂,制备^(68)Ga-L-NETA-四嗪(Tz)分子探针,测定其标记率、稳定性。体外培养基底样乳腺癌细胞MDA-MB-468(EGFR+)和MDA-MB-231(EGFR-),进行细胞摄取及阻断实验。用Balb/c-nu小鼠建立MDA-MB-468和MDA-MB-231皮下荷瘤鼠模型;将50μg Cetuximab-TCO注入荷瘤鼠体内,经过不同时间(48、36、24和12 h)后再注射^(68)Ga-L-NETA-Tz,利用"TCO-Tz"反应(点击化学特异性结合)实现^(68)Ga-L-NETA-Tz与Cetuximab-TCO的体内连接;进行小动物PET显像及生物分布实验。数据比较采用单因素方差分析。结果:成功制备^(68)Ga-L-NETA-Tz分子探针,标记率>95%,2 h放化纯>95%。体外细胞摄取实验证实了预定位技术的可行性:先后加入Cetuximab-TCO与^(68)Ga-L-NETA-Tz后,1 h时MDA-MB-468细胞摄取率可达(0.69±0.04)%。体内PET显像及生物分布实验结果示,提前36 h注射Cetuximab-TCO,MDA-MB-468荷瘤鼠有最高的肿瘤摄取值[(0.77±0.05)每克组织百分注射剂量率(%ID/g),1 h]及最佳的靶/非靶比值(肿瘤/肌肉:4.67±0.46);给予过量Cetuximab的阻断组、未提前注入Cetuximab-TCO组及MDA-MB-231组肿瘤未见明显摄取[(0.35±0.01)、(0.39±0.05)、(0.45±0.10)%ID/g;F=15.50,P=0.002]。结论:采用预定位技术,通过先后注射Cetuximab-TCO和^(68)Ga-L-NETA-Tz,在荷瘤鼠模型中成功进行了靶向EGFR免疫PET显像,为单抗免疫PET显像提供了一种有效的方法。Objective To explore the feasibility of pretargeting technique for immunoPET with epidermal growth factor receptor(EGFR)monoclonal antibody in EGFR positive/negative tumor bearing mice.Methods Cetuximab-Trans-cyclooctene(TCO)was obtained by modifying Cetuximab with TCO-N-hydroxysuccinimide(NHS).2,2′-((6-amino-1-(4,7-bis-(carboxymethyl)-1,4,7-triazonan-1-yl)hexan-2-yl)azanediyl)-diacetic acid(L-NETA)was used as a chelating agent to prepare the radioligand ^(68)Ga-L-NETA-tetrazine(Tz),then the labeling rate and in vitro stability of the product were determined.Human basal breast cancer cells MDA-MB-468(EGFR+)and MDA-MB-231(EGFR-)were cultured in vitro.In vitro experiments were performed to explore the specificity of the probe and the feasibility of pretargeting technique.Nude mice(Balb/c-nu)bearing xenografts of the above two cell lines were established.Cetuximab-TCO(50μg)was injected into the tumor-bearing mice in advance,then ^(68)Ga-L-NETA-Tz was injected at different time points(48,36,24 and 12 h),and pretargeting was realized through"click chemistry".Small-animal PET imaging and biodistribution were performed to evaluate pharmacokinetic properties and specificity of the probe.The one-way analysis of variance was used to compare the data.Results The ^(68)Ga-L-NETA-Tz molecular probe was successfully prepared with the labeling yield>95%,and the radiochemical purity was>95%after 2 h.Cetuximab-TCO and ^(68)Ga-L-NETA-Tz were added to MDA-MB-468 cells successively,and the cell uptake rate reached(0.69±0.04)%at 1 h,which demonstrated the feasibility of the pretargeting technique.PET imaging and biodistribution results showed that the best imaging results were obtained in 36 h pre-injection group,in which the tumor uptake was the highest((0.77±0.05)percentage activity of injection dose per gram of tissue(%ID/g),1 h)and the tumor/muscle ratio was optimal(4.67±0.46);the tumor uptake in the blocking group,the group without injecting Cetuximab-TCO,and the MDA-MB-231 group were significantly lower((0.35±0.01),(0.39�

关 键 词：抗体 单克隆 受体 表皮生长因子 点击化学 同位素标记 镓放射性同位素 正电子发射断层显像术 乳腺肿瘤 小鼠 裸 

分 类 号：R817.4[医药卫生—影像医学与核医学]