Preclinical Study of Carbonic Anhydrase IX and Prostate-Specific Membrane Antigen Bispecific Probe for Synergistic Targeting of Hypoxia and Neovasculature

The heterogeneous expression of tumor biomarkers limits the diagnostic performance of single-target imaging agents. Carbonic anhydrase IX (CAIX) is highly expressed in hypoxic regions of clear cell renal cell carcinoma (ccRCC) and multiple solid tumors, whereas prostate-specific membrane antigen (PSMA) is specifically upregulated in tumor-associated neovasculature. Both targets have been implicated in tumor metastasis and poor clinical outcomes. This study aimed to design and evaluate a novel bispecific PET tracer, [⁶⁸Ga]Ga-PCA, targeting both CAIX and PSMA, with the goal of achieving improved tumor-specific uptake. Subcutaneous xenograft models were established in nude mice by inoculation with OS-RC-2, PC3-PIP, and HEK-293 cells. PET/CT imaging and biodistribution studies were performed following intravenous administration of [⁶⁸Ga]Ga-PCA. Target specificity was evaluated via competitive blocking assays employing excess unlabeled ligand. Immunohistochemical staining was performed to validate the expression profiles of the targets within the tumors. After being labeled with gallium-68, [⁶⁸Ga]Ga-PCA showed favorable physicochemical properties, such as a high radiolabeling yield (>80%), radiochemical purity over 95%, good stability in vitro, and an albumin-binding rate of 93.44 ± 0.81%. PET/CT imaging revealed pronounced and specific tracer accumulation in both OS-RC-2 and PC3-PIP tumor models. In OS-RC-2 tumors (PSMA⁺/CAIX⁺), the SUVmax (13.10 ± 0.84) was higher than those of the single-target tracers [⁶⁸Ga]Ga-DOTA-NY104 (5.31 ± 0.77) and [⁶⁸Ga]Ga-PSMA (2.31 ± 0.49) at 60 min postinjection. An excess of unlabeled DOTA-NY104, a PSMA-targeted ligand, or a mix of the two ligands can block the uptake of [⁶⁸Ga]Ga-PCA. These results demonstrate that the tracer can bind to both targets at once. In conclusion, [⁶⁸Ga]Ga-PCA is a bispecific PET tracer that targets both hypoxic tumor cells and tumor neovasculature by binding to both CAIX and PSMA. The probe exhibited significant specificity, advantageous imaging contrast, and robust blocking validation, indicating its potential for molecular imaging of malignancies, including clear cell renal cell carcinoma (ccRCC).