A heterobivalent claudin 18.2/FAP dual-target PET probe enables enhanced imaging of gastric cancer

Claudin 18.2 (CLDN18.2)-positive tumor epithelial compartments and fibroblast activation protein (FAP)-positive stroma enriched in cancer-associated fibroblasts (CAFs) often exhibit complementary spatiotemporal heterogeneity, which may lead to unstable lesion detection or insufficient contrast when using single-target molecular imaging. We aimed to develop a FAP/CLDN18.2 dual-target probe, [ 68 Ga]Ga-DOTA-FZ2R, and to evaluate imaging performance in comparison with single-target probes. A dual-target radiotracer, [ 68 Ga]Ga-DOTA-FZ2R, targeting CLDN18.2 and FAP, was designed and synthesized. Its targeting ability and specificity were evaluated in CLDN18.2-positive and FAP-positive cells and tumor models (n = 3 per group or time point). Small-animal positron emission tomography/computed tomography (PET/CT) was performed in single-positive and dual-positive models with comparison against the corresponding single-target tracers. CLDN18.2 and FAP expression were also analyzed in a cohort of 34 patients with stage III poorly differentiated gastric cancer to assess their correlation and explore potential prognostic associations. [ 68 Ga]Ga-DOTA-FZ2R showed high radiochemical purity, good stability, and strong affinity for both targets. In single-positive models, it achieved higher tumor uptake, longer tumor retention, and higher tumor-to-muscle and tumor-to-liver ratios than the corresponding monomeric tracers; notably, tumor uptake was more than 4-fold higher than that of [68Ga]Ga-FAPI-04. In dual-positive models (KPC1199CLDN18.2 and NUGC4CLDN18.2), the observed tumor uptake exceeded the predefined additive reference calculated from the two single-target tracers under the study conditions. Moreover, in the patient cohort, CLDN18.2 and FAP expression were independent (R2 = 0.002, P = 0.79), and combined assessment provided hypothesis-generating observations regarding potential prognostic stratification. [ 68 Ga]Ga-DOTA-FZ2R may enable simultaneous targeting of tumor epithelial and stromal compartments and may improve imaging performance in preclinical models with complementary CLDN18.2/FAP heterogeneity. These findings support the feasibility of a dual-target PET imaging strategy for gastric cancer and warrant further clinical PET/CT validation.