Development of Novel Peptide-Based Radiotracers for Detecting FGL1 Expression in Tumors

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 68 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 50 ranging from 100 nM to 160 nM. Among the probes, PET imaging revealed that 68 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 68 Ga-NODA-FGLP21 was significantly higher than those of 68 Ga-NODA-FGLP22 and 68 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 68 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 68 Ga-NODA-FGLP21 has the potential to specifically detect FGL1 expression in tumors with good contrast to the background.