A Trifunctional Theranostic Ligand Targeting Fibroblast Activation Protein-α (FAPα)

Fibroblast activation protein-α (FAPα) is uniquely expressed in activated fibroblasts, including cancer-associated fibroblasts that populate tumor stroma and contribute to proliferation and immunosuppression. Radiolabeled FAPα inhibitors enable imaging of multiple human cancers, but time-dependent clearance from tumors currently limits their utility as FAPα-targeted radiotherapeutics. We sought to increase the area under the curve (AUC) by constructing a trifunctional ligand that binds FAPα with high affinity and also binds albumin and theranostic radiometals.

RPS-309 comprised a FAPα-targeting moiety, an albumin-binding group, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Inhibition of recombinant human FAPα (rhFAPα) was determined by colorimetric assay. Affinity for human serum albumin (HSA) was determined by high-performance affinity chromatography. The tissue distribution of [⁶⁸Ga]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 [¹⁷⁷Lu]Lu-RPS-309 was determined at 4, 24, and 96 h p.i.

RPS-309 inhibits rhFAPα with IC₅₀ = 7.3 ± 1.4 nM. [⁶⁸Ga]Ga-RPS-309 is taken up specifically by FAPα-expressing cells and binds HSA with K_d = 4.6 ± 0.1 μM. 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) × h.

RPS-309 is a high-affinity FAPα inhibitor with prolonged plasma residence. Introduction of the albumin-binding group did not compromise FAPα binding. Although initial tumor uptake was high and FAPα-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.