Radiosynthesis and characterization of a 68Ga-labeled uPAR-targeting peptide

Objectives The increased expression the urokinase plasminogen activator receptor (uPAR) in many cancer types is associated with establishment of malignant disease, proliferation and invasiveness. Interest in uPAR has intensified as its role in regulating many cancers has become clear, leading to the development of peptide-based agents that target uPAR for diagnostic and therapeutic approaches. In this study, we report the radiolabeling of a high-affinity uPAR-targeting peptide with generator-produced 68Ga, optimization of labeling conditions, and stability.

Methods A linear uPAR-targeting peptide (AE105) was synthesized and conjugated to DOTA on solid support to yield U1. 68Ga was eluted form a commercially-available generator and the eluate was either fractionated or concentrated by cation exchange. Radiolabeling was performed in sodium acetate buffer at pH 4 and heated at 95oC. Reaction conditions were optimized for heating time, peptide concentration, and buffer concentration (0.1 M or 1.25 M). Stability of the radiolabeled complex was assessed in PBS, DTPA and mouse serum.

Results Radiolabeling and purification of 68Ga-U1 resulted in radiochemical purity >99%. Dilute sodium acetate showed lower labeling yields, thus all subsequent reactions were carried out in 1.25 M sodium acetate. Longer heating improved complexation of 68Ga and reached the maximum incorporation yield at 10 mins. The impact of peptide amount was shown by low labeling yields with <10 nmol of the agent, whereas 80-92% labeling could be achieved when using 20-30 nmol. The agent proved very stable in all conditions with minimal presence of free 68Ga at 3 hrs incubation.

Conclusions A robust scheme for synthesis and purification of 68Ga-U1 was established and radiolabeling conditions were optimized. The excellent in vitro stability of the agent coupled with the favorable characteristics of 68Ga warrant pharmacological evaluation in uPAR-expressing cells