Development of CSF1R-Targeted Peptide Tracers for PET Imaging of LPS-Induced Pulmonary Inflammation

Abstract The colony-stimulating factor 1 receptor (CSF1R) is a highly promising target for the noninvasive imaging of macrophage-driven inflammatory diseases. To develop a targeted molecular tracer, we first discovered a high-affinity peptide ligand (CP, KD = 0.188 μM) toward CSF1R using mRNA display. We then designed and synthesized a series of DOTA-conjugated peptides (CP0-CP4) featuring systematically extended hydrophilic (2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) linkers. Upon 68Ga-labeling, linker elongation progressively enhanced the tracer’s hydrophilicity and dramatically improved its serum stability (intact fraction at 60 min increased from 12.0% for 68Ga-CP0 to 84.1% for 68Ga-CP4). The optimized candidate, 68Ga-CP4, demonstrated specific binding and internalization in CSF1R-overexpressing cells. In normal mice, increasing linker length effectively reduced nonspecific uptake and promoted rapid renal clearance. In a murine model of LPS-induced pulmonary inflammation, 68Ga-CP4 clearly visualized inflamed lungs, with significantly higher uptake (SUV 2.40 ± 0.39 at 60 min) compared to sham controls (1.81 ± 0.10). Specificity was confirmed by effective blockade with unlabeled peptide (SUV 0.89 ± 0.16) and ex vivo biodistribution. CSF1R protein upregulation in inflamed lungs was further validated by immunohistochemical staining and Western blot. This work presents 68Ga-CP4 as a novel, peptide-based PET tracer for imaging CSF1R-positive inflammation.