Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of68Ga-Labeled FAPI Dimer

Cancer-associated fibroblasts (CAFs) are crucial components of the tumor microenvironment. Fibroblast activation protein (FAP) is overexpressed in CAFs. FAP-targeted molecular imaging agents, including FAP inhibitor (FAPI)-04 and FAPI-46, have shown promising results in tumor diagnosis. However, these molecules have relatively short tumor-retention time for peptide-targeted radionuclide therapy applications. We aimed to design a ⁶⁸Ga-labeled FAPI dimer (denoted as ⁶⁸Ga-DOTA-2P(FAPI)2) to optimize the pharmacokinetics and evaluate whether this form is more effective than its monomeric analogs. Methods:⁶⁸Ga-DOTA-2P(FAPI)2 was synthesized based on the quinoline-based FAPI variants (FAPI-46), and its binding properties were assayed in CAFs. Preclinical pharmacokinetics was determined in FAP-positive patient-derived xenografts (PDXs) using small-animal PET and biodistribution experiments. The effective dosimetry of ⁶⁸Ga-DOTA-2P(FAPI)2 was evaluated in three healthy volunteers, and PET/ CT imaging of ⁶⁸Ga-FAPI-46 and ⁶⁸Ga-DOTA-2P(FAPI)2 was performed in three cancer patients. Results:⁶⁸Ga-DOTA-2P(FAPI)2 was stable in phosphate-buffered saline and fetal bovine serum for 4 h. The FAPI dimer showed high affinity and specificity for FAP in-vitro and in-vivo. The tumor uptake of ⁶⁸Ga-DOTA-2P(FAPI)2 was approximately two-fold stronger than that of ⁶⁸Ga-FAPI-46 in PDXs, while the healthy organs showed low tracer uptake and fast body clearance. The effective dose of ⁶⁸Ga-DOTA-2P(FAPI)2 was 1.19E-02 mSv/MBq, calculated using OLINDA. Finally, PET/CT scans in three cancer patients revealed higher intratumoral uptake of ⁶⁸Ga-DOTA-2P(FAPI)2 than that of ⁶⁸Ga-FAPI-46 in all tumor lesions (maximum standardized uptake value: 8.1-39.0 vs. 1.7-24.0, respectively; P < 0.001). Conclusion:⁶⁸Ga-DOTA-2P(FAPI)2 has increased tumor uptake and retention properties compared to ⁶⁸Ga-FAPI-46, and it could be a promising tracer for both diagnostic imaging and targeted therapy of malignant tumors with positive expression of FAP.