^(124)I标记抗体及抗体片段用于胃癌动物模型表皮生长因子受体表达水平

Non-invasive evaluation of epidermal growth factor receptor expression in gastric cancer animal models using^(124)I-labeled antibody and antibody fragment

西妥昔单抗(Cetuximab)常用于晚期胃癌肿瘤患者的治疗,获得患者肿瘤表皮生长因子受体(Epidermal growth factor receptor,EGFR)的整体表达水平对于预测西妥昔单抗的疗效尤为重要。本研究拟通过对比研究的方式筛选最符合临床需求的靶向EGFR的探针。本研究基于西妥昔单抗通过酶切法制备了Cet-F(ab')_(2)和Cet-Fab,并偶联CY3-NHS酯(Cyanine 3 NHS ester)得CY3-Cetuximab、CY3-Cet-F(ab')_(2)和CY3-Cet-Fab进行细胞荧光实验以验证亲和力。通过Iodogen(1,3,4,6-四氯-3α,6α-二苯基甘脲)法制备得到3种^(124)I标记的分子探针:^(124)I-Cetuximab、^(124)I-Cet-F(ab')_(2)和^(124)I-Cet-Fab,使用micro-PET/CT分别在探针注射后1 h、3 h、6 h、12 h显像进行对比研究。最后,通过生物学分布实验探究3种探针在各时间点在体内的分布情况,采用两独立样本t检验进行组间比较。西妥昔单抗经过片段化后,亲和力无显著降低。Cetuximab、Cet-F(ab')_(2)和Cet-Fab分子量分别约为150 kDa、100 kDa和50 kDa。经^(124)I标记并纯化后,^(124)I-Cetuximab、^(124)I-Cet-F(ab')_(2)和^(124)I-Cet-Fab放射化学纯度均大于94%,比活度均约为2×10^(4) MBq/μmol。^(124)I-Cetuximab、^(124)I-Cet-F(ab')_(2)和^(124)I-Cet-Fab在生理盐水和胎牛血清(FBS)中稳定性好,体外放置24 h放射化学纯度仍高于90%。Micro-PET/CT显像显示在^(124)I-Cet-Fab尾静脉注射后3 h肿瘤部位明显浓聚,^(124)I-Cet-F(ab')_(2)尾静脉注射后12 h肿瘤部位明显浓聚,而^(124)I-Cetuximab尾静脉注射后12 h肿瘤部位仍无明显显像剂分布。生物学分布结果与micro-PET/CT结果一致。3种正电子探针中,^(124)I-Cet-Fab代谢速度最快,肿瘤最早显象的同时有最少的累积辐射剂量,有一定的临床转化潜力。

Cetuximab is commonly used to treat patients with advanced gastric cancer and the overall level of tumor epidermal growth factor receptor(EGFR)expression critically impacts the efficacy of cetuximab.This study was performed to select the most clinically relevant probes targeting EGFR via comparative research.Cetuximab was fragmented by enzymatic cleavage to produce Cet-F(ab')_(2)and Cet-Fab,which were then conjugated with CY3 NHS ester to obtain CY3-Cetuximab,CY3-Cet-F(ab')_(2),and CY3-Cet-Fab for cell fluorescence experiments to verify their affinity.Three^(124)I-labeled molecular probes,namely^(124)I-Cetuximab,^(124)I-Cet-F(ab')_(2),and^(124)I-Cet-Fab,were prepared using the Iodogen method.Micro-PET/CT imaging was performed 1 h,3 h,6 h,and 12 h after probe injection for comparative studies.Further,biological distribution experiments were conducted to explore the in vivo distributions of the three probes at different time points.Independent sample t-tests were used for intergroup comparisons.Fragmentation did not significantly decrease cetuximab affinity.The respective molecular weights of Cetuximab,Cet-F(ab')_(2),and Cet-Fab were approximately 150 kDa,100 kDa,and 50 kDa,respectively.After^(124)I labeling and purification,the radiochemical purity of^(124)I-Cetuximab,^(124)I-Cet-F(ab')_(2),and^(124)I-Cet-Fab exceeded 94%,and the specific activity was approximately 2×10^(4)MBq/μmol.Stability tests in saline and fetal bovine serum(FBS)showed that radiochemical purity remained above 90%after 24 h of incubation.Micro-PET/CT imaging demonstrated significant accumulation of^(124)I-Cet-Fab at the tumor site 3 h after intravenous injection,whereas^(124)ICet-F(ab')_(2)exhibited pronounced accumulation at the tumor site 12 h after injection.However,no evident distribution of the imaging agent was observed at the tumor site 12 h after intravenous injection of^(124)I-Cetuximab.The biological distribution results are consistent with the micro-PET/CT findings.Among the three positron probes,^(124)I-Cet-Fab exhibited the fastest metabolism,earliest tumor imaging,and the lowest cumulative radiation dose,indicating its potential for clinical translation.