ImmunoPET imaging of c-Met using a nanobody-based tracer [68Ga]Ga-NOTA-PFCM01 in pancreatic ductal adenocarcinoma models and non-human primates.

Abstract

PURPOSE Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer, with high malignancy and poor prognosis. The cellular mesenchymal-epithelial transition factor (c-Met) is overexpressed in 84% of PDAC and plays a critical role in tumor progression, which is closely associated with poor patient outcomes. In this study, a new 68Ga-labeled nanobody ([68Ga]Ga-NOTA-PFCM01) was developed for the visualization of c-Met in PDAC models. METHODS The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) data were utilized to assess MET expression and overall survival in patients with different cancers. By immunizing an alpaca with recombinant human c-Met, three clones of nanobodies were screened, and the binding affinity was tested by bio-layer interferometry (BLI). The binding epitope of the nanobodies and c-Met was predicted by AlphaFold3. c-Met expression in human PDAC cell lines was evaluated using western blot, flow cytometry, and confocal microscopy. NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) chelator was used to label the nanobodies with 68Ga. PET imaging, semiquantitative uptake, and biodistribution research were carried out in tumor xenografts. Histological staining was performed on tumor tissues to characterize the expression of c-Met. In addition, PET imaging in non-human primates was performed to assess pharmacokinetics and biodistribution of the nanobody. RESULTS Based on TCGA and GTEx data, MET expression in pancreatic adenocarcinoma (PAAD) is significantly higher than that in normal tissues (P < 0.001). Patients with high MET expression have lower overall survival rates than those with low MET expression. c-Met expression was the highest in BXPC-3 cells but the lowest in MIA PaCa-2 cells, which were set as the positive and negative models respectively. PFCM01 was screened and selected with an excellent binding property with the KD value of 0.16 nM. The radiochemical purity (RCP) of [68Ga]Ga-NOTA-PFCM01 was high, with good in vivo and in vitro stability. Semiquantitative analysis of small animal positron emission tomography/computed tomography (PET/CT) imaging demonstrated significantly higher tumor uptake in BXPC-3 tumors at 2 h post-injection (2.31 ± 0.02%ID/g) than control groups. Ex vivo biodistribution also showed a high uptake of BXPC-3 tumors (1.90 ± 0.59%ID/g) than others, which was further verified the PET imaging results. Histological staining showed high expression of c-Met in BXPC-3 but low in MIA PaCa-2 tumor tissues. In healthy cynomolgus monkeys, PET imaging revealed rapid renal excretion of [68Ga]Ga-NOTA-PFCM01. No significant radioactive uptake was observed in the liver, stomach, intestines, pancreas, or muscles, indicating its favorable pharmacokinetics and translational potential. CONCLUSIONS [68Ga]Ga-NOTA-PFCM01 showed a specific and high tumor uptake in c-Met-positive PDAC models, providing a noninvasive method to assess c-Met expression. Besides, it demonstrated good pharmacokinetic characteristics in non-human primates. Therefore, [68Ga]Ga-NOTA-PFCM01 provided the potential role as a prospective PET imaging agent for future clinical applications.