Development and Preclinical Evaluation of [68Ga]Ga-DOTA-L3: A Novel PET Tracer for Quantitative Imaging of Carbonic Anhydrase IX

Abstract Carbonic anhydrase IX (CA IX) is a key biomarker for tumor hypoxia and a promising target for molecular imaging. In this study, we developed [68Ga]Ga-DOTA-L3, a novel small-molecule CA IX-targeted PET tracer. The radiolabeling was optimized to consistently achieve a radiochemical purity of >98% (10 μg precursor, 90 °C, pH 4.5, 10 min), with excellent stability in saline and FBS up to 240 min. In vivo studies demonstrated rapid blood clearance and predominant renal excretion. Surface plasmon resonance analysis demonstrated high-affinity binding of the DOTA-L3 precursor toward CA IX (Kd = 7.60 ± 0.37 nM) with highest affinity observed for CA IX among the tested isoforms. MicroPET/CT imaging (30−240 min p.i.) showed robust tumor accumulation in OSRC-2 (SUVmean = 1.47 ± 0.21) and HT-29 (SUVmean = 1.03 ± 0.12) xenografts, while minimal uptake was observed in CA IX-low FTC-238 tumors (SUVmean = 0.21 ± 0.05) at 60 min p.i. PET-derived tumor uptake showed a strong linear correlation with CA IX expression levels (R2 = 0.976, P < 0.001) across the three tumor models. High tumor-to-muscle and tumor-to-blood ratios further confirm favorable imaging contrast (T/M = 27.53 ± 1.89 and T/B = 24.61 ± 2.35 at 240 min p.i. for OSRC-2 model). These results support [68Ga]Ga-DOTA-L3 as a promising candidate for non-invasive assessment of CA IX-associated hypoxia and as a scaffold for future theranostic development.