靶向成纤维细胞激活蛋白四聚体放射性探针的构建及其在荷瘤裸鼠中的诊疗一体化研究

Development of radiolabeled tetramer that targeting fibroblast activation protein and theranostic research in tumor xenografts

目的制备靶向成纤维细胞激活蛋白(FAP)的四聚体探针1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(DOTA)-4P[FAP抑制剂(FAPI)]4,评价其在FAP表达阳性荷瘤裸鼠模型中的生物分布和PET显像,并探讨其用于FAP阳性肿瘤治疗的可行性。方法基于FAPI-46单体结构在4个FAPI活性基团之间各加入4个微型聚乙二醇(PEG)(PEG_(3)),构建靶向FAP的四聚体探针4P(FAPI)4,并经螯合剂DOTA偶联完成四聚体探针的^(68)Ga和^(177)Lu标记。通过体外细胞实验鉴定四聚体在体外对FAP结合性能。在FAP表达阳性的HT-1080-FAP荷瘤裸鼠模型中进行小动物PET显像和生物分布实验,并行核素靶向内照射治疗的实验研究(共39只)。肿瘤摄取数据比较行两独立样本t检验分析;核素治疗中肿瘤体积数据的比较采用重复测量方差分析。结果细胞体外实验显示,DOTA-4P(FAPI)4和单体FAPI-46的半数抑制浓度值相似(3.29和2.15 nmol/L)。PET显像及生物分布数据显示,^(68)Ga/^(177)Lu标记的FAPI四聚体具有高度靶向FAP的特异性,且较其单体FAPI-46具有更高的肿瘤摄取及更持久的瘤内滞留时间:注射后48 h^(177)Lu-DOTA-4P(FAPI)4和^(177)Lu-FAPI-46肿瘤摄取分别为(18.72±1.32)与(2.72±1.20)每克组织百分注射剂量率(%ID/g)(t=15.55,P<0.001)。在核素治疗实验中,^(177)Lu-DOTA-4P(FAPI)4较^(177)Lu-FAPI-46和对照组(100μl生理盐水注射)显示出更好的抗肿瘤治疗效果:在治疗开始后第2天,四聚体治疗组肿瘤体积明显小于对照组(平均值差值67.19 mm^(3),P=0.049);在治疗开始后第14天,四聚体治疗组肿瘤体积明显小于单体治疗组(平均值差值414.33 mm^(3),P=0.005)。结论与FAPI-46相比,FAPI四聚体DOTA-4P(FAPI)4在肿瘤摄取和滞留方面均有较大幅度的提升,^(177)Lu-DOTA-4P(FAPI)4有望成为一种有前景的放射性药物应用于FAP阳性肿瘤的核素内照射治疗。

Objective To develop a tetramer probe targeting fibroblast activation protein(FAP),named 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA)-4P(FAP inhibitor(FAPI))4,evaluate its biodistribution and PET image in FAP-positive-tumor bearing nude mice,and explore its feasibility as a novel radio-regent for treatment of FAP-positive tumor.Methods FAP tetramer probe was constructed on the FAPI-46 motif with four mini-polyethylene glycol(PEG)(PEG3)spacers between the four FAPI motifs,denoted as 4P(FAPI)4.DOTA was used as the chelator for radiolabeling with ^(68)Ga and ^(177)Lu.The FAP binding characteristics were test by in vitro cell competitive binding experiment.Small-animal PET,in vivo biodistribution,and radionuclide targeting therapy were performed in HT-1080-FAP tumor bearing nude mice(n=39).Independent-sample t test was performed to analyze tumor uptake data,and two-factor repeated measures analysis of variance was utilized to compare tumor volume data in radioactive isotope therapy.Results Cell experiment showed that FAPI-tetramer and FAPI-monomer had similar half maximal inhibitory concentration values(3.29 and 2.15 nmol/L).^(68)Ga/^(177)Lu radiolabeled FAPI-tetramer had better tumor uptake and retention than FAPI-monomer in small-animal PET and in vivo biodistribution experiment,with the tumor uptake for ^(177)Lu-DOTA-4P(FAPI)4 and ^(177)Lu-FAPI-46 at 48 h of(18.72±1.32)vs(2.72±1.20)percentage activity of injection dose per gram of tissue(%ID/g)(t=15.55,P<0.001).^(177)Lu-DOTA-4P(FAPI)4 group showed best anti-tumor efficacy compared with ^(177)Lu-FAPI-46 and control group in radionuclide targeting therapy.On the 2nd day after the start of treatment,the tumor volume in the tetramer treatment group was significantly smaller than that in the control group(mean difference 67.19 mm^(3),P=0.049);on the 14th day after the start of treatment,the tumor volume in the tetramer treatment group was significantly smaller than that in the monomer treatment group(mean difference 414.33 mm^(3),P=0.005).Conclusion FAPI-tetramer can improve tumor uptake and retention ability compared with FAPI-46,and ^(177)Lu-DOTA-4P(FAPI)4 can be a promising radio-agent for FAP-positive tumor therapy.