Xu Yue, Zhang Jinyuan, Pan Donghui, Yan Junjie, Chen Chongyang, Wang Lizhen, Wang Xinyu, Yang Min, Xu Yuping
School of Pharmacy, Nanjing Medical University, Nanjing 211166, China.
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China.
Mol Pharm. 2025 Mar 3;22(3):1605-1614. doi: 10.1021/acs.molpharmaceut.4c01293. Epub 2025 Feb 2.
A novel immune checkpoint, FGL1, is a potentially viable target for tumor immunotherapy. The development of FGL1-targeted PET probes could provide significant insights into the immune system's status and the evaluation of treatment efficacy. A ClusPro 2.0 server was used to analyze the interaction between FGL1 and LAG3, and the candidate peptides were identified by using the Rosetta peptide derivate protocol. Three candidate peptides targeting FGL1, named FGLP21, FGLP22, and FGLP23, with a simulated affinity of -9.56, -8.55, and -8.71 kcal/mol, respectively, were identified. The peptides were readily conjugated with p-NCS-benzyl-NODA-GA, and the resulting compounds were successfully labeled with Ga in approximately 70% yields and radiochemical purity greater than 95%. In vitro competitive cell-binding assay demonstrated that all probes bound to FGL1 with IC ranging from 100 nM to 160 nM. Among the probes, PET imaging revealed that Ga-NODA-FGLP21 exhibited the best tumor imaging performance in mice bearing FGL1 positive Huh7 tumor. At 60 min p.i., the tumor uptake of Ga-NODA-FGLP21 was significantly higher than those of Ga-NODA-FGLP22 and Ga-NODA-FGLP23, respectively (2.51 ± 0.11% ID/g vs 1.00 ± 0.16% ID/g and 1.49 ± 0.05% ID/g). Simultaneously, the tumor-to-muscle uptake ratios of the former were also higher than those of the latter, respectively (19.40 ± 2.30 vs 9.65 ± 0.62 and 12.45 ± 0.72). In the presence of unlabeled FGLP21, the uptake of Ga-NODA-FGLP21 in Huh7 xenograft decreased to 0.81 ± 0.09% ID/g at 60 min p.i., which is similar to that observed in the FGL1 negative U87 MG tumor (0.46 ± 0.03% ID/g). The results were consistent with the immunohistochemical analysis and ex vivo autoradiography. No significant radioactivity was accumulated in normal organs, except for kidneys. In summary, a preclinical study confirmed that the tracer Ga-NODA-FGLP21 has the potential to specifically detect FGL1 expression in tumors with good contrast to the background.
一种新型免疫检查点FGL1是肿瘤免疫治疗的潜在可行靶点。开发靶向FGL1的PET探针可为了解免疫系统状态和评估治疗效果提供重要见解。使用ClusPro 2.0服务器分析FGL1与LAG3之间的相互作用，并使用Rosetta肽衍生方案鉴定候选肽。鉴定出三种靶向FGL1的候选肽，分别命名为FGLP21、FGLP22和FGLP23，模拟亲和力分别为-9.56、-8.55和-8.71 kcal/mol。这些肽很容易与对硝基苯硫氰酸苄基-NODA-GA偶联，所得化合物以约70%的产率成功用镓标记，放射化学纯度大于95%。体外竞争性细胞结合试验表明，所有探针均以100 nM至160 nM的IC与FGL1结合。在这些探针中，PET成像显示，Ga-NODA-FGLP21在携带FGL1阳性Huh7肿瘤的小鼠中表现出最佳的肿瘤成像性能。在注射后60分钟时，Ga-NODA-FGLP21的肿瘤摄取量分别显著高于Ga-NODA-FGLP22和Ga-NODA-FGLP23（2.51±0.11% ID/g对1.00±0.16% ID/g和1.49±0.05% ID/g）。同时，前者的肿瘤与肌肉摄取比也分别高于后者（19.40±2.30对9.65±0.62和12.45±0.72）。在未标记的FGLP21存在下，注射后60分钟时，Huh7异种移植瘤中Ga-NODA-FGLP21摄取量降至0.81±0.09% ID/g，与FGL1阴性U87 MG肿瘤中观察到的摄取量相似（0.46±0.03% ID/g）。结果与免疫组织化学分析和离体放射自显影一致。除肾脏外，正常器官中未积累明显放射性。总之，一项临床前研究证实，示踪剂Ga-NODA-FGLP21有潜力特异性检测肿瘤中FGL1的表达，与背景有良好对比度。
