Background: To enhance tracer's efficacy, multimerization of fibroblast activation protein (FAP)- targeted probes have shown superior performance. In this study, a novel N-acetylgalactosamine (GalNAc)-linker-based FAPI trimer probe was designed and synthesized to improve tumor targeting affinity and retention. Methods: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-FAPI-FUSCC-NAC (DOTA-FAPI-FUSCC-NAC) was radiolabeled with both 68 Ga and 177 Lu. In vitro evaluations, including stability, binding affinity, hydrophilicity/lipophilicity, plasma protein binding, and cell uptake, were conducted. Furthermore, positron emission tomography/computed tomography (PET/CT) imaging was performed in tumor-bearing mice. Pharmacokinetics, stability in vivo , and ex vivo distribution studies were conducted to assess the probe's imaging performance and tumor-targeting capabilities. In addition, 177 Lu-DOTA-FAPI-FUSCC-NAC relevant tests were performed to evaluate its potential as a treatment tracer. Results: DOTA-FAPI-FUSCC-NAC was successfully radiolabeled with both 68 Ga and 177 Lu. Comprehensive in vitro and in vivo evaluations demonstrated that DOTA-FAPI-FUSCC-NAC exhibits an extremely high binding affinity for FAP, with an IC 50 of 0.426 nmol/L, 8 times greater than that of FAPI-04 (3.5 nmol/L). Additionally, 68 Ga-DOTA-FAPI-FUSCC-NAC showed high tumor specificity and significantly prolonged retention in FAP-positive tumors. PET/CT imaging studies showed that 68 Ga-DOTA-FAPI-FUSCC-NAC provided clear, sustained imaging signals with a high tumor to non-target tissue (T/NT) ratio, while biodistribution studies confirmed efficient clearance from non-target tissues. Moreover, the 177 Lu-labeled FAPI trimer demonstrated substantial tumor uptake, indicating potential for therapeutic applications. Conclusion: These findings suggest that the GalNAc-linker-based FAPI trimer probe offers a promising advancement in FAP-targeted imaging and theranostics, with implications for improved diagnosis and treatment of FAP-associated diseases.
Cheng et al. (Mon,) studied this question.