Kelly_2021_Mol.Imaging.Biol_23_686

PURPOSE: Fibroblast activation protein-alpha (FAPalpha) is uniquely expressed in activated fibroblasts, including cancer-associated fibroblasts that populate tumor stroma and contribute to proliferation and immunosuppression. Radiolabeled FAPalpha inhibitors enable imaging of multiple human cancers, but time-dependent clearance from tumors currently limits their utility as FAPalpha-targeted radiotherapeutics. We sought to increase the area under the curve (AUC) by constructing a trifunctional ligand that binds FAPalpha with high affinity and also binds albumin and theranostic radiometals. PROCEDURES: RPS-309 comprised a FAPalpha-targeting moiety, an albumin-binding group, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Inhibition of recombinant human FAPalpha (rhFAPalpha) was determined by colorimetric assay. Affinity for human serum albumin (HSA) was determined by high-performance affinity chromatography. The tissue distribution of [(68)Ga]Ga-RPS-309 in SW872 tumor xenograft-bearing mice was imaged by microPET/CT and quantified by biodistribution studies performed from 30 min to 3 h post injection (p.i.). The biodistribution of [(177)Lu]Lu-RPS-309 was determined at 4, 24, and 96 h p.i. RESULTS: RPS-309 inhibits rhFAPalpha with IC(50) = 7.3 +/- 1.4 nM. [(68)Ga]Ga-RPS-309 is taken up specifically by FAPalpha-expressing cells and binds HSA with K(d) = 4.6 +/- 0.1 microM. Uptake of the radiolabeled ligand in tumors was evident from 30 min p.i. (> 5 %ID/g) and was significantly reduced by co-injection of RPS-309. Specific skeletal uptake was also observed. Activity in tumors was constant through 4 h p.i., but cleared significantly by 24 h. The AUC in this period was 127 (%ID/g) x h. CONCLUSIONS: RPS-309 is a high-affinity FAPalpha inhibitor with prolonged plasma residence. Introduction of the albumin-binding group did not compromise FAPalpha binding. Although initial tumor uptake was high and FAPalpha-specific, RPS-309 also progressively cleared from tumors. Nevertheless, RPS-309 incorporates multiple sites in which structural diversity can be introduced, and therefore serves as a platform for future structure-activity relationship studies.