Efficacy of targeted radionuclide therapy Using 131 I-FAPi dimers labeled via tetrazine ligation in a human glioblastoma xenograft model

The successful use of diagnostic radiotracers targeting the pan-tumor marker fibroblast activation protein (FAP) across numerous cancer types is well established; however, translating these agents into effective therapeutics remains challenging. Compared with monomeric FAP inhibitors (FAPi), FAPi dimers-containing two FAP-targeting vectors-exhibit enhanced tumor retention, resulting in higher tumor doses and encouraging preliminary clinical outcomes. Current therapeutic agents targeting FAP rely largely on chelator-based constructs, restricting their labeling to radiometals. To overcome this limitation, we previously developed FAPi dimers equipped for tetrazine ligation using the novel trans-cyclooctene T4CO, enabling selective formation of a single isomer. Our lead compound, (¹³¹I)I-FAPi 5, was synthesized with high yield and purity and evaluated in tumor-bearing animals, where it demonstrated therapeutic effects comparable to the established agent (¹⁷⁷Lu)Lu-DOTAGA-Glu(FAPi)₂. These findings suggest that (¹³¹I)I-FAPi 5 may offer a cost-effective and scalable radioligand therapy (RLT) option to meet the expanding demand for FAP-directed treatments. (¹³¹I)I-DUAL FAPi-5 was prepared by a two-step radiolabeling process using tetrazine ligation. First, a tetrazine-trimethylgermyl precursor was radioiodinated with ¹³¹I. The resulting synthon was purified by HPLC and subsequently clicked with the T4CO-FAPi derivative, achieving complete ligation within 10 minutes without requiring further purification. The therapeutic effect was investigated in U87-MG tumor-bearing mice. When the tumors reached 64±4mm 3 , the mice received a single intravenous injection of ( 131 I)I-FAPi 5 (30 or 60 MBq), ( 177 Lu)Lu-DOTAGA-Glu(FAPi)2 (30 MBq) or vehicle control (n=6/group). Tumor response was monitored every other day via caliper measurement and survival was assessed up to 70 days after the start of treatment. The mice were euthanized on reaching one of the predefined endpoint criteria.(¹³¹I)I-FAPi 5 was obtained in two steps with a final radiochemical yield of 60 ± 5% (n=4), radiochemical purity ≥98% (n=4). In U87-MG xenograft models, the tracer exhibited high and sustained tumor uptake. Treatment with (¹³¹I)I-FAPi 5 at both dose levels (30 MBq and 60 MBq) resulted in rapid tumor regression, with therapeutic performance comparable to that of (¹⁷⁷Lu)Lu-DOTAGA-Glu(FAPi)₂. Our dual-targeting FAPi compound labeled with ¹³¹I demonstrated robust tumor uptake and retention, with renal clearance as the primary excretion pathway. These findings confirm the therapeutic efficacy of this agent in preclinical tumor models, showing marked tumor regression and improved survival. Collectively, the results support the potential of this compound as an effective and accessible FAP-RLT candidate.