Site-specific development and preclinical evaluation of 68Ga-THP-ACN376, a CLDN18.2-targeted nanobody probe

Objective: To construct a Claudin 18.2 (CLDN18.2)-targeted positron emission tomography (PET) probe, 68Ga-THP-ACN376, using a site-specific labeling technique, and to evaluate its in vitro and in vivo biological performance. Methods: The chelator THP-Mal was site-specifically conjugated to the CLDN18.2-targeting nanobody ACN376 to synthesize the precursor compound THP-ACN376, which was subsequently radiolabeled with 68Ga. The labeling efficiency, radiochemical purity, and in vitro stability of the probe were determined by radio thin layer chromatography (Radio-TLC). Micro-PET/computed tomography (CT) imaging was performed in BALB/c nude mice to assess its in vivo pharmacokinetic properties and targeting capability. Results: 68Ga-THP-ACN376 exhibited high labeling efficiency (>99%), high radiochemical purity (>99%), and a high specific activity of 16 GBq/μmol. It remained stable in saline, 5% human serum albumin (HSA), and phosphate buffered saline (PBS). Micro-PET/CT results demonstrated that the metabolism of the probe in mice followed the typical profile of nanobodies, with tracer uptake in the stomach gradually increasing from 0.5 to 2 h. The maximum standardized uptake value SUVmax in the stomach at 2 h was 1.35±0.07. Conclusion: This study successfully constructed a CLDN18.2-targeted PET probe, 68Ga-THP-ACN376, using a site-specific labeling method. The probe demonstrates high labeling efficiency, remarkable stability, and significant targeting ability, showing great potential for detecting CLDN18.2 protein expression levels.