In vitro and in vivo evaluation of targeted probe 68 Ga-NOTA-376 constructed by non-site-specific labeling method for CLDN18.2

Objective: To construct a positron emission tomography (PET) probe, 68 Ga-NOTA-376, targeting CLDN18.2 using a non-site-specific labeling method, and to evaluate its in vitro and in vivo properties. Methods: The chelating agent p-SCN-Bn-NOTA was used to non-site-specifically conjugate the nanobody 376 targeting CLDN18.2, resulting in the precursor NOTA-376, which was then radiolabeled with 68 Ga. The labeling efficiency, radiochemical purity, and in vitro stability of the probe were determined by radio- thin layer chromatography (Radio-TLC), and micro-PET/ computed tomography (CT) imaging experiments were conducted in a nude mouse model of human gastric adenocarcinoma. Results: 68 Ga-NOTA-376 demonstrated a high labeling yield (89.98±0.07) %, high radiochemical purity (97.67±0.02)% and high specific activity (16.69±6.60) GBq/μmol, and remained stable in 5% human serum albumin (HSA) and phosphate-buffered saline (PBS). Micro-PET/CT results indicated that the maximum standardized uptake value (SUV max ) of the probe in CLDN18.2-positive tumors was significantly higher than in CLDN18.2-negative tumors. At 4.0 h, the SUV max of CLDN18.2- positive tumors and CLDN18.2-negative tumors were 9.08±0.33 and 1.92±0.32, respectively. Conclusion: This study successfully constructed a CLDN18.2-targeting probe 68 Ga-NOTA-376 using a non-site-specific labeling method, which showed high labeling efficiency, good stability, and targeting capability, making it a potential PET probe for the detection of CLDN18.2 protein expression levels.