[${}^{64}$Cu]Cu-FAP-NOX, a N-oxalyl modified cyclic peptide for FAP PET imaging with a flexible imaging time window

The aim of the present study was to develop a novel ${}^{64}$Cu-labeled cyclic peptide ([${}^{64}$Cu]Cu-FAP-NOX) that targets fibroblast activation protein (FAP) and may offer advantages in terms of image contrast, imaging time window, and low uptake in normal tissues. The novel cyclic peptide featuring with a N-oxalyl modified tail was constructed and conjugated to NOTA for ${}^{64}$Cu labeling. Biochemical and cellular assays were performed with A549.hFAP cells. The performance of [${}^{64}$Cu]Cu-FAP-NOX was compared to that of two established tracers ([${}^{64}$Cu]Cu-FAPI-04 and [${}^{68}$Ga]Ga-FAP-2286) and three different NOTA-conjugates in HEK-293T.hFAP xenograft mice using micro-PET imaging. Ex vivo biodistribution studies were performed to confirm the FAP specificity and to validate the PET data. Furthermore, a first-in-human study of this novel tracer was conducted on one patient with lung cancer. Compared to [${}^{64}$Cu]Cu-FAPI-04, [${}^{64}$Cu]Cu-FAP-NOX demonstrated faster and higher rates of cellular uptake and internalization in A549.hFAP cells, but lower rates of cellular efflux. All six radiotracers were rapidly taken up by the tumor within the first 4 h post-injection. However, [${}^{64}$Cu]Cu-FAP-NOX had more intense tumor accumulation and slower washout from the target. The ratios of the tumor to normal tissue (including kidneys and muscles) increased significantly over time, with [${}^{64}$Cu]Cu-FAP-NOX reaching the highest ratio among all tracers. In the patient, [${}^{64}$Cu]Cu-FAP-NOX PET showed a comparable result to FDG PET in the primary malignant lesion while exhibiting higher uptake in pleural metastases, consistent with elevated FAP expression as confirmed by immunohistochemistry. [${}^{64}$Cu]Cu-FAP-NOX is a promising FAP-targeted tracer with a highly flexible imaging time window, as evidenced by preclinical evaluation encompassing biodistribution and micro-PET studies, along with a successful patient application. Furthermore, [${}^{64}$Cu]Cu-FAP-NOX showed enhanced image contrast and favorable pharmacokinetic properties for FAP PET imaging, warranting translation into large cohort studies.