A ⁶⁸Ga/¹⁶¹Tb-labeled FAP-targeted radioligand for enhanced theranostics: Prolonged tumor retention and metastasis...

Fibroblast activation protein (FAP) is a promising theranostic target in stroma-rich malignancies. However, conventional FAP inhibitors exhibit rapid tumor washout, limiting their application in targeted radionuclide therapy. To address this limitation, we rationally designed two novel FAP-targeted radioligands, FAPI-RuiJ01 and FAPI-RuiJ02, by integrating glutathione or cysteine as hydrophilic linkers, respectively. In preclinical evaluations, FAPI-RuiJ01 showed sub-nanomolar FAP binding affinity (IC₅₀ = 0.55 nM), enhanced hydrophilicity, prolonged tumor retention, low hepatosplenic uptake, and favorable tumor-to-background contrast. FAPI-RuiJ02 also exhibited FAP-specific uptake and improved hydrophilicity, however, its tumor uptake, retention and dosimetric profile were not consistently superior to those of FAPI-04 and were less favorable than those of FAPI-RuiJ01. In a preliminary first-in-human positron emission tomography/computed tomography (PET/CT) study of patients with gastric cancer (n = 5 per group), ⁶⁸Ga-FAPI-RuiJ01 and ⁶⁸Ga-FAPI-RuiJ02 showed higher uptake than ⁶⁸Ga-FAPI-04 in metastatic lesions (P < 0.05), whereas in primary lesions, higher numerical values were observed without reaching statistical significance. For therapeutic evaluation, the tracers were labeled with Terbium-161 (¹⁶¹Tb), a radionuclide that emits both β- particles and Auger electrons. ¹⁶¹Tb-FAPI-RuiJ02 did not significantly inhibit tumor growth and showed therapeutic efficacy comparable to that of ¹⁶¹Tb-FAPI-04. ¹⁶¹Tb-FAPI-RuiJ01 suppressed primary tumor growth and reduced the lung metastatic burden in an orthotopic 4T1 breast cancer model. Mechanistically, ¹⁶¹Tb-FAPI-RuiJ01 induced substantial DNA double-strand breaks in vitro, and in vivo it promoted tumor cell death while suppressing proliferation. Transcriptomic and flow cytometric profiling further revealed that this treatment was associated with the enrichment of interferon-related signaling, along with remodeling of the immune microenvironment. Collectively, these findings support FAPI-RuiJ01 as a promising FAP-targeted theranostic radioligand that warrants further evaluation in metastatic malignancies.