Rational design and preclinical evaluation of ⁶⁸Ga-labeled PEG-modified dimeric FAP radiotracers

Fibroblast activation protein (FAP), which is highly overexpressed on cancer-associated fibroblasts (CAFs) while being minimally expressed in normal adult tissues, has emerged as an attractive target for tumor imaging and radionuclide therapy. In this study, we report the design and synthesis of a series of PEG-modified dimeric FAP-targeted radiotracers ([⁶⁸Ga]Ga-HBEDFAP-H1 to [⁶⁸Ga]Ga-HBEDFAP-H4) based on an HBED-CC chelation scaffold and UAMC-1110-derived FAP-binding motifs, with the aim of systematically modulating tumor uptake and in vivo pharmacokinetics. Micro-PET/CT imaging in U87 tumor-bearing nude mice revealed a pronounced dependence of in vivo behavior on PEG linker length. Among the series, [⁶⁸Ga]Ga-HBEDFAP-H3 exhibited the highest tumor uptake (SUV_max = 4.11 ± 0.23 at 1 h p.i.), highlighting the strong tumor-targeting capability conferred by the dimeric design. Further PEG linker extension produced [⁶⁸Ga]Ga-HBEDFAP-H4, which showed moderately reduced tumor uptake (SUV_max = 1.98 ± 0.59 at 1 h p.i.) but substantially decreased hepatic and renal accumulation, leading to prolonged tumor retention and improved tumor-to-background contrast at later time points. Blocking studies confirmed FAP-specific tumor uptake. Notably, compared with the monomeric tracer [⁶⁸Ga]Ga-FAPI-04 (SUV_max = 0.29 ± 0.11 at 1 h p.i.), [⁶⁸Ga]Ga-HBEDFAP-H4 exhibited markedly enhanced tumor uptake and retention. Collectively, these results demonstrate that the HBED-CC-based dimerization strategy, combined with rational PEGylation, effectively improves the in vivo performance of FAP-targeted tracers and supports their further development for FAP-directed theranostic applications.