PD-L1靶向单域抗体的定点标记及生物学评价  

Site-specific labelling and biological evaluation of PD-L1 targeted single-domain antibody

作　　者：陈银飞 陈盼盼 朱诗宇 梁蓓蓓 谢敏浩[1] 林建国[1] 邱玲[1] Chen Yinfei;Chen Panpan;Zhu Shiyu;Liang Beibei;Xie Minhao;Lin Jianguo;Qiu Ling(NHC Key Laboratory of Nuclear Medicine,Jiangsu Key Laboratory of Molecular Nuclear Medicine,Jiangsu Institute of Nuclear Medicine,Wuxi 214063,China)

机构地区：[1]国家卫生健康委员会核医学重点实验室、江苏省分子核医学重点实验室、江苏省原子医学研究所,无锡214063

出　　处：《中华核医学与分子影像杂志》2022年第12期744-750,共7页Chinese Journal of Nuclear Medicine and Molecular Imaging

基　　金：无锡市科技发展资金项目(Y20212013)。

摘　　要：目的设计合成^(68)Ga-1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(DOTA)-半胱氨酸-天冬氨酸-缬氨酸(CDV)-Nb109,探讨其用于检测不同肿瘤中程序性细胞死亡蛋白配体1(PD-L1)表达水平的潜力。方法采用基因工程技术将CDV引入单域抗体Nb109序列的尾部,通过马来酰亚胺-半胱氨酸定点偶联策略,将马来酰亚胺-DOTA与CDV-Nb109(物质的量比1∶1)反应制备前体DOTA-CDV-Nb109,随后进行^(68)Ga标记并用PD-10脱盐柱纯化。建立人黑色素瘤A375、人源性PD-L1转染的A375-hPD-L1及人胶质瘤U87荷瘤裸鼠模型,通过稳定性分析、细胞摄取和microPET显像评价^(68)Ga-DOTA-CDV-Nb109的诊断价值。采用单因素方差分析和最小显著差异t检验分析数据。结果^(68)Ga-DOTA-CDV-Nb109放射化学产率为(69.79±4.69)%,放化纯大于97%,摩尔活度为(12.85±1.51)GBq/μmol。^(68)Ga-DOTA-CDV-Nb109与A375-hPD-L1细胞具有较强的亲和力,解离常数(K_(d))为(66.43±17.89)nmol/L。^(68)Ga-DOTA-CDV-Nb109在A375-hPD-L1细胞[(3.17±0.15)百分加入放射性剂量(%AD)]、U87细胞[(2.08±0.03)%AD]中的摄取明显高于A375细胞[(1.21±0.14)%AD;F=82.87,t值:15.23、9.98,P值:<0.001、0.003]。^(68)Ga-DOTA-CDV-Nb109在A375-hPD-L1[(5.21±0.35)每毫升百分注射剂量率(%ID/ml)]和U87[(3.44±0.69)%ID/ml]荷瘤裸鼠肿瘤中的摄取显著高于A375荷瘤裸鼠[(2.17±0.36)%ID/ml;F=249.72,t值:35.70、3.43,均P<0.001]。结论成功制得定点标记的PET探针^(68)Ga-DOTA-CDV-Nb109,可无创、动态监测不同肿瘤中PD-L1表达水平的变化,在PD-L1免疫检查点阻断治疗潜在受益患者筛选方面具有应用潜力。Objective To synthesize a novel site-specifically labelled probe^(68)Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA)-Cys-Asp-Val(CDV)-Nb109 and explore its potential for detection of the programmed cell death ligand 1(PD-L1)expression level in different tumors.Methods Firstly,CDV was inserted into the tail of the sequence of Nb109 by genetic engineering.Then the precursor DOTA-CDV-Nb109 was prepared by mixing the maleimide-DOTA and the single-domain antibody CDV-Nb109(amount of substance ratio 1∶1)via the maleimide-cysteine site-specific coupling strategy.Subsequently,the DOTA-CDV-Nb109 was labeled with^(68)Ga and purified by PD-10 column.Human melanoma A375,human PD-L1 transfected melanoma A375-hPD-L1 and human glioma U87 tumor-bearing mice models were established,and the diagnostic value of^(68)Ga-DOTA-CDV-Nb109 was evaluated by stability assay,cellular uptake,and microPET imaging.One-way analysis of variance and the least significant difference t test were used to analyze the data.Results The probe^(68)Ga-DOTA-CDV-Nb109 was obtained with the radiochemical yield of(69.79±4.69)%,radiochemical purity more than 97%,and molar activity of(12.85±1.51)GBq/μmol.^(68)Ga-DOTA-CDV-Nb109 had strong binding affinity for A375-hPD-L1 with the dissociation constant(K_(d))of(66.43±17.89)nmol/L.The uptake of^(68)Ga-DOTA-CDV-Nb109 in A375-hPD-L1 and U87 cells were(3.17±0.15)percentage of the added radioactivity dose(%AD)and(2.08±0.03)%AD respectively,which were significantly higher than that in A375 cells((1.21±0.14)%AD;F=82.87,t values:15.23,9.98,P values:<0.001,0.003).The tumor uptake of the probe in A375-hPD-L1((5.21±0.35)percentage of injected dose per ml(%ID/ml))and U87 tumor-bearing mice((3.44±0.69)%ID/ml)were significantly higher than that in A375 tumor-bearing mice((2.17±0.36)%ID/ml;F=249.72,t values:35.70,3.43,both P<0.001).Conclusion The site-specifically labelled probe^(68)Ga-DOTA-CDV-Nb109,which can non-invasively and dynamically monitor the change of PD-L1 expression level in different t

关 键 词：程序性细胞死亡受体1 单域抗体 同位素标记 镓放射性同位素 肿瘤细胞 培养的 正电子发射断层显像术 小鼠 裸 

分 类 号：R392-33[医药卫生—免疫学]