177Lu标记HER2亲和体的制备及性能研究

Preparation and properties of 177Lu-labeled HER2 affibody

目的:制备一种177Lu标记的人表皮生长因子受体2(HER2)亲和体177Lu-1,4,7-三氮杂环壬烷-1,4,7-三乙酸(NOTA)-马来酰亚胺(Mal)-半胱氨酸(Cys)-ZHER 2:342(简称177Lu-NOTA-MZHER2),探讨其标记工艺及抗肿瘤性能。方法:考察2种缓冲液体系(乙酸钠缓冲液体系和抗坏血酸钠缓冲液体系),比较pH值、前体质量与反应温度对177Lu标记NOTA-MZHER2的影响,获取最佳标记条件。采用快速薄层色谱(ITLC)测定标记产物放化纯,观察其在PBS和血浆中的稳定性。取人源性卵巢癌细胞SKOV-3进行细胞内化和细胞毒性实验,评价177Lu-NOTA-MZHER2的细胞摄取和杀伤效果。取SKOV-3荷瘤鼠(n=3)注射177Lu-NOTA-MZHER2后进行microSPECT/CT显像。另取荷瘤鼠40只,分为尾静脉注射探针22.2 MBq(静注22.2 MBq)组、对照组(尾静脉注射PBS)、低剂量组(瘤体注射探针3.7 MBq)和高剂量组(瘤体注射探针7.4 MBq),每组10只,注射探针后监测肿瘤体积和荷瘤鼠体质量,评估标记产物的抗肿瘤效应和毒性。采用重复测量方差分析(Bonferroni法)比较数据间的差异。结果:乙酸钠缓冲液体系下,pH=4、前体质量50μg、70~80℃反应30 min,为最优标记条件。在此条件下,标记产物177Lu-NOTA-MZHER2的标记率为(99.3±0.4)%,放化纯>99%;在PBS和血浆中放置12 d后,放化纯分别为(95.0±1.5)%和(95.0±2.1)%。细胞实验结果显示,177Lu-NOTA-MZHER2的细胞内化占总摄取的(29.02±3.50)%,在标记产物放射性浓度为6×10-3 Bq/L时,SKOV-3细胞的存活率为(48±6)%。SPECT显像示,注射18.5 MBq 177Lu-NOTA-MZHER2后96 h,该药仍在肿瘤部位浓聚。静注22.2 MBq组、高剂量组、低剂量组与对照组比较,荷瘤鼠的相对肿瘤体积(RTV)差异有统计学意义(F=21.75,P<0.001);高剂量组注射后20 d,荷瘤鼠RTV为(140±7)%,相对体质量为(80±9)%,与对照组相比,具有明显的抗肿瘤效果(均P<0.001)。结论:成功制备177Lu-NOTA-MZHER2,工艺简单高效,该药具有较好的抗肿瘤效果。

Objective To prepare a 177Lu labeled human epidermal growth factor receptor 2(HER2)affibody 177Lu-1,4,7-triazacyclononane-1,4,7-triacetic acid(NOTA)-maleimide(Mal)-cysteine(Cys)-ZHER2:342(177Lu-NOTA-MZHER2 for short),and investigate its labeling process and anti-tumor properties.Methods Two kinds of buffer systems(sodium acetate buffer system and sodium ascorbate buffer system)were investigated.The effects of pH value,precursor mass and reaction temperature on 177Lu labeling NOTA-MZHER2 were compared to obtain optimal labeling conditions.The radiochemical purity of labeled product was determined by instant thin-layer chromatography(ITLC),and its stabilities in PBS and plasma were observed.Human ovarian cancer cell line SKOV-3 was selected for cell internalization and cytotoxicity test to evaluate cell uptake and killing effect of 177Lu-NOTA-MZHER2.SKOV-3 tumor-bearing mice(n=3)were injected with 177Lu-NOTA-MZHER2,and microSPECT/CT imaging was performed.Another 40 tumor-bearing mice were divided into 22.2 MBq group(tail vein injection with probe of 22.2 MBq),control group(tail vein injection with PBS),low-dose group(tumor injection with probe of 3.7 MBq)and high-dose group(tumor injection with probe of 7.4 MBq).Tumor volume and mass of tumor-bearing mice were monitored after injection,and the anti-tumor effect and toxicity of probe were evaluated.Repeated measurement analysis of variance(Bonferroni method)was used to analyze the data.Results The optimal labeling condition was 70-80℃for 30 min in the system of sodium acetate buffer solution with pH=4 and precursor mass of 50μg.Under these conditions,the labeling rate of 177Lu-NOTA-MZHER2 was(99.3±0.4)%and radiochemical purity was>99%.After 12 d in PBS and plasma,the radiochemical purities were(95.0±1.5)%and(95.0±2.1)%.Results of cell experiment showed that the internalization of 177Lu-NOTA-MZHER2 accounted for(29.02±3.50)%of the total uptake,and the survival rate of SKOV-3 cells was(48±6)%with the probe concerntration of 6×10-3 Bq/L.SPECT imaging showed that 177Lu-NOTA-MZHER2 was still concentrated at the tumor site 96 h after injection with a dose of 18.5 MBq.Relative tumor volume(RTV)of tumor-bearing mice in 22.2 MBq group,high-dose group and low-dose group was significantly different from that in control group(F=21.75,P<0.001).Twenty days after injection,RTV and relative body mass of the tumor-bearing mice in high-dose group were(140±7)%and(80±9)%,respectively.Compared with control group,high-dose group had obvious anti-tumor effect(both P<0.001).Conclusion 177Lu-NOTA-MZHER2 is successfully prepared,which is simple and efficient,and the probe has good anti-tumor effect.