Systematic PEGylation optimization of Dar3A-based FAP-targeted PET probes and first-in-human study

Bifunctional chelators are essential for the development of novel radiopharmaceuticals. For the 68Ga-based radiopharmaceuticals, previously reported chelator 2-[11,27-bis(carboxymethyl)-34,36-dihydroxy-7,23-dimethyl-3,11,19,27,33,35-hexaazapentacyclo[27.3.1.15,9.113,17.121,25]hexatriaconta-1(33),5(36),6,8,13,15,17(35),21(34),22,24,29,31-dodecaen-3-yl]acetic acid (Dar3A) demonstrated high labeling efficiency, while the corresponding FAP-targeted probe exhibited enhanced tumor uptake. Still, its utility was limited by excessive hepatic accumulation and increased background signals. To attenuate the high hepatic uptake and optimize in vivo pharmacokinetics, a series of Dar3A-based ligands incorporating hydrophilic polyethylene glycol (PEG) chains of varying lengths was designed and synthesized. The five PEGylated Dar3A-PEG ligands were synthesized by inserting PEG chains of varying lengths as linkers between the Dar3A chelator and the fibroblast activation protein (FAP)-targeting motif. The theoretical binding interactions were simulated using molecular docking. Preclinical evaluations consisted of PET imaging and biodistribution in U87MG tumor-bearing mice and pharmacokinetic studies in BALB/c mice. Subsequently, human PET studies were performed using the lead candidate to assess its clinical translational potential. The five 68Ga-labeled FAPI probes showed favorable affinity and in vitro stability. Among the evaluated candidates, [68Ga]Ga-Dar3A-P5-F showed the highest tumor uptake in U87MG tumor-bearing mice, with tumor uptake reaching 7.40 ± 1.54 %ID/g at 2 h and further increasing to 9.90 ± 0.52 %ID/g at 4 h. Furthermore, primary tumors and various metastatic lesions in cancer patients were clearly delineated by [68Ga]Ga-Dar3A-P5-F in the human PET studies. [68Ga]Ga-Dar3A-P5-F was identified as the most promising candidate among the evaluated PEGylated probes. The clinical translation potential of the lead probe was further supported.