Squaric-acid-engineered FAP2286: A next-generation ⁶⁸Ga/¹⁷⁷Lu theranostic ligand with synergistically enhanced...

To systematically enhance the theranostic performance of the FAP-targeted ligand FAP2286 by rational chemical modification-introducing methoxy (MeO) and squaric acid (SA) substituents-followed by comparative ⁶⁸Ga/¹⁷⁷Lu radiolabeling and pre-clinical validation.

An integrated workflow combined in-silico (molecular docking, 100-ns GROMACS MD), in-vitro (IC₅₀, LogP, plasma-protein binding, radiochemical stability, cell uptake/release), and in-vivo (U87MG and HEK-293-FAP xenografts; micro-PET/CT, micro-SPECT/CT; biodistribution; single-dose ¹⁷⁷Lu-radiotherapy) evaluations.

SA conjugation conferred the highest FAP affinity (docking score - 9.475 kcal mol^-1; IC₅₀ 2.94 ± 0.41 nM), greatest hydrophilicity (LogP -5.27 ± 0.06), and > 95 % radiochemical integrity after 48 h in serum. ⁶⁸Ga-FAP2286-SA achieved rapid tumor uptake (23.4 ± 2.1 %ID g^-1 at 4 h) with T/B = 78.0; ¹⁷⁷Lu-FAP2286-SA displayed sustained accumulation (23.5 ± 2.1 %ID g^-1 at 48 h) and T/B = 33.6. A single 37 MBq dose of ¹⁷⁷Lu-FAP2286-SA produced 186.0 % TGI (day 33) without overt hepatorenal or hematologic toxicity. MeO substitution modestly reduced hepatic background yet significantly compromised tumor retention and therapeutic efficacy.

Squaric acid derivatization synergistically elevates affinity, hydrophilicity, and intratumoral residence while preserving safety, establishing ⁶⁸Ga/¹⁷⁷Lu-FAP2286-SA as a clinically translatable, next-generation theranostic pair for FAP-positive malignancies.