Development and comparison of 68Ga/18F/64Cu-labeled nanobody tracers probing Claudin18.2

Claudin 18.2 (CLDN18.2) is an emerging target for the treatment of gastric cancers. We aim to develop tracers to image the expression of CLDN18.2. A humanized nanobody targeting CLDN18.2 (clone hu19V3) was produced and labeled with Ga, Cu, and F. The tracers were investigated in subcutaneous and metastatic models established using two different mouse types (nude and Balb/c mice) and two different cell lines (CHO-CLDN18.2 and CT26-CLDN18.2). Gastric cancer patient-derived xenograft (PDX) models were further established for validation experiments. Three novel CLDN18.2-targeted tracers (i.e., [Ga]Ga-NOTA-hu19V3, [Cu]Cu-NOTA-hu19V3, and [F]F-hu19V3) were developed with good radiochemical yields and excellent radiochemical purities. [Ga]Ga-NOTA-hu19V3 immuno-positron emission tomography (immunoPET) rapidly delineated subcutaneous CHO-CLDN18.2 lesions and CT26-CLDN18.2 tumors, as well as showing excellent diagnostic value in PDX models naturally expressing CLDN18.2. While [Ga]Ga-NOTA-hu19V3 had high kidney accumulation, [Cu]Cu-NOTA-hu19V3 showed reduced kidney accumulation and improved image contrast at late time points. Moreover, [F]F-hu19V3 was developed via click chemistry reaction under mild conditions and precisely disseminated CHO-CLDN18.2 lesions in the lungs. Furthermore, region of interest analysis, biodistribution study, and histopathological staining results correlated well with the imaging results. Taken together, immunoPET imaging with the three tracers can reliably visualize CLDN18.2 expression.