Development of FAPI Tetramers to Improve Tumor Uptake and Efficacy of FAPI Radioligand Therapy

Radiolabeled fibroblast activation protein (FAP) inhibitors (FAPIs) have shown promise as cancer diagnostic agents; however, the relatively short tumor retention of FAPIs may limit their application in radioligand therapy. In this paper, we report the design, synthesis, and evaluation of a FAPI tetramer. The aim of the study was to evaluate the tumor-targeting characteristics of radiolabeled FAPI multimers in vitro and in vivo, thereby providing information for the design of FAP-targeted radiopharmaceuticals based on the polyvalency principle. Methods: FAPI tetramers were synthesized on the basis of FAPI-46 and radiolabeled with ⁶⁸Ga, ⁶⁴Cu, and ¹⁷⁷Lu. In vitro FAP-binding characteristics were identified using a competitive cell-binding experiment. To evaluate their pharmacokinetics, small-animal PET, SPECT, and ex vivo biodistribution analyses were performed on HT-1080-FAP and U87MG tumor–bearing mice. In addition, the 2 tumor xenografts received radioligand therapy with ¹⁷⁷Lu-DOTA-4P(FAPI)₄, and the antitumor efficacy of the ¹⁷⁷Lu-FAPI tetramer was evaluated and compared with that of the ¹⁷⁷Lu-FAPI dimer and monomer. Results:⁶⁸Ga-DOTA-4P(FAPI)₄ and ¹⁷⁷Lu-DOTA-4P(FAPI)₄ were highly stable in phosphate-buffered saline and fetal bovine serum. The FAPI tetramer exhibited high FAP-binding affinity and specificity both in vitro and in vivo. ⁶⁸Ga-, ⁶⁴Cu-, and ¹⁷⁷Lu-labeled FAPI tetramers exhibited higher tumor uptake, longer tumor retention, and slower clearance than FAPI dimers and FAPI-46 in HT-1080-FAP tumors. The uptake (percentage injected dose per gram) of ¹⁷⁷Lu-DOTA-4P(FAPI)₄, ¹⁷⁷Lu-DOTA-2P(FAPI)₂, and ¹⁷⁷Lu-FAPI-46 in HT-1080-FAP tumors at 24 h was 21.4 ± 1.7, 17.1 ± 3.9, and 3.4 ± 0.7, respectively. Moreover, ⁶⁸Ga-DOTA-4P(FAPI)₄ uptake in U87MG tumors was approximately 2-fold the uptake of ⁶⁸Ga-DOTA-2P(FAPI)₂ (SUVmean, 0.72 ± 0.02 vs. 0.42 ± 0.03, P < 0.001) and more than 4-fold the uptake of ⁶⁸Ga-FAPI-46 (0.16 ± 0.01, P < 0.001). In the radioligand therapy study, remarkable tumor suppression was observed with the ¹⁷⁷Lu-FAPI tetramer in both HT-1080-FAP and U87MG tumor–bearing mice. Conclusion: The satisfactory FAP-binding affinity and specificity, as well as the favorable in vivo pharmacokinetics of the FAPI tetramer, make it a promising radiopharmaceutical for theranostic applications. Improved tumor uptake and prolonged retention of the ¹⁷⁷Lu-FAPI tetramer resulted in excellent characteristics for FAPI imaging and radioligand therapy.