Validation of a Ready-to-Use Lyophilized Kit for Labeling IL2 with (68)Ga: A New Avenue for Imaging Activated T-lymphocytes in Tumor Microenvironment.

Background/Objectives: Radiolabeled interleukin-2 (IL2) could allow for imaging activated T-lymphocytes in the tumor microenvironment (TME). The aims of this study were to assess the shelf life of a lyophilized kit containing THP-desIL2 to allow for the labeling of IL2 with (68)Ga at room temperature and to test the in vitro binding of (68)Ga-THP-desIL2 on different T-cell populations in order to determine which specific T-cell subset expresses the CD25 subunit of the IL2 receptor (IL2R). Methods: desIL2 was conjugated with THP and lyophilized. (68)Ga labeling was performed and several quality controls, including HPLC, iTLC and SDS-PAGE, were carried out at different storage times (1, 3 and 6 months) and temperatures (4 °C and -80 °C). Moreover, flow cytometric analysis on different T-cell populations and the in vitro and competitive binding of (68)Ga-THP-desIL2 were performed. Results: The lyophilized kit of THP-desIL2 was stable up to 6 months at -80 °C, preserving its sterility, integrity and acceptable values of labeling yield (51.80 ± 3.74%), radiochemical purity (>96%) and specific activity (5.59 ± 0.40 MBq/µg). Binding of (68)Ga-THP-desIL2 on activated lymphocytes was specific and exhibited a low dissociation constant from IL2R on stimulated Tregs (Kd: 10(-9)-10(-10) mol/L). Conclusions: We assessed the shelf life of a lyophilized kit containing THP-desIL2 for the easy labeling of IL2 with (68)Ga at room temperature. The kit can be stored at -80 °C up to 6 months, thus facilitating the adoption of (68)Ga-THP-desIL2 into clinical practice. (68)Ga-THP-desIL2 showed high affinity and specificity for CD25 on activated T-lymphocytes, particularly Tregs, thus opening new opportunities for imaging immune cells trafficking in the TME.