PET/CT imaging of the peptide-based anti-CD47 probe 18FAlF-NOTA-Pep-20 in preclinical models

Cluster of differentiation 47 (CD47), a key immune checkpoint, is significantly overexpressed in tumors and transmits a 'don't eat me' signal that facilitates tumor immune evasion. This study aims to develop a peptide-based positron emission tomography/computed tomography (PET/CT) tracer specific for CD47 and assessed its diagnostic potential in preclinical models. We synthesized a cysteine-modified, NOTA-conjugated Pep-20 (NOTA-Pep-20) and assessed its binding affinity for CD47 via molecular docking and surface plasmon resonance (SPR). The fluorine-18-labeled probe, 18FAlF-NOTA-Pep-20, was characterized in vitro for stability and specificity. Biodistribution and imaging performance were then evaluated in several tumor-bearing animal models. Molecular docking and SPR analyses confirmed the moderate affinity of NOTA-Pep-20 for CD47. In vitro assays demonstrated both the high CD47 specificity of 18FAlF-NOTA-Pep-20 and its significant cellular internalization. Subcutaneous tumors were clearly visualized via PET/CT imaging just one hour after probe injection. An in vivo blocking study and ex vivo biodistribution analysis further validated the specific CD47-targeting ability of 18FAlF-NOTA-Pep-20. Dynamic PET/CT imaging in C6 tumor-bearing rats revealed rapid renal clearance of the probe, which yielded low background signals. In conclusion,this study reports the first development of a peptide-based anti-CD47 probe, 18FAlF-NOTA-Pep-20, and confirms its feasibility and safety for visualizing CD47 expression.