Imaging activated T-lymphocytes in chronic inflammatory diseases and in tumor micro-environment, is a task that cannot be successfully accomplished ex-vivo with histological techniques (due to limitations of bioptic material) or in-vivo with conventional radiological techniques (like computed tomography, ultrasonography or nuclear magnetic resonance). On the other hand, monitoring activated T-cells in vivo has become a priority to define indication and to monitor the efficacy of immunotherapies (i.e., immune check point inhibitors).
Interleukin-2 (IL2) is a cytokine mainly produced by T-cells with an important role in lymphocyte growth and differentiation. IL2 binds to specific receptors (CD25, CD122 and CD132), mainly expressed on activated T-lymphocytes (cytotoxic T-cells and T-regs).
Several studies have been published with 123I-IL2 (1-3), 99mTc-IL2 (4-6) and 18F-IL2 (7,8), all highlighting the important clinical role of this imaging modality. Nevertheless, none of the above radiopharmaceutical is easily and cheaply synthetized, thus limiting its clinical use.
Our purpose was to develop a ready to use kit to label IL2 with 68Ga for non-invasively imaging activated T-cell trafficking by PET/CT.
Proleukin (recombinant IL2-Aldesleukin®-Novartis, desIL2) was chemically modified with a bifunctional chelating agent, the tris-hydroxypyridinone (THP), to allow the radiolabelling with 68Ga at room temperature. The THP-IL2 was purified by 24h dialysis, freeze dried in aliquots and stored at -80°C. HPLC and mass-spectroscopy were performed to identify how many molecules of THP were bound to IL2 and in which aminoacid residue. Radiolabelling was performed with GAIA synthesis module in GMP at room temperature for 30’ followed by tC2 purification to eliminate unbound 68Ga. HPLC and ITLC analysis were then performed to calculate radiochemical yield (RCY%), radiochemical purity (RCP%), specific activity (SA) and molar activity (MA).
For in vitro binding assay, lymphocytes were collected from several donors and stimulated with PHA or with IL2 and negatively sorted by FACS to obtain different cell subsets (CD4+, CD8+, CD25+, B-cells, NK and Treg). Pre-clinical studies, in 12 healthy BALB/c mice, were performed to evaluate the 68Ga-IL2 biodistribution at different time points (15’, 1h, 2h) after injection of 3.7 MBq (100µCi, 1µg 68Ga-IL2). Blood and major organs were collected and the percentage of injected dose per organ (%ID) and per gram (%ID/g) were calculated.
For phase I human study, 159.6±15.2MBq of 68Ga-IL2 (4.31±0.41mCi; 26.8±2.5µg 68Ga-IL2) were administered i.v. in 5 volunteers and total body PET/CT images acquired at 15’, 30’, 60’, 90’, 120’, 150’ and 210’ to assess biodistribution and dosimetry (IDAC-Dose 2.1).
Mass spectrometry of THP-IL2 showed that more than 70% of the IL2 was conjugated with only 1 molecule of THP. QCs of 68Ga-THP-IL2 showed: RCY of 53.67±10.4%, RCP of 97.91±0.45%, SA of 5.94±0.42MBq/µg and MA 90.95±6.44GBq/µmol.
In vitro binding on PHA-activated PBMCs showed a Kd=10-9-10-10 M. Similar results were obtained on stimulated Treg cells, B-cells and NK cells. Slightly lower Kd (10-8 M) was measured for unstimulated CD8+ cells and CD4+ cells.
68Ga-IL2 showed excellent biodistribution in mice, both in terms of %ID/g and %ID, with a rapid and predominant kidney metabolism. At 1h and 2h post injection, the spleen had the highest %ID/g, as expected for the physiological presence of activated T-cells.
Similar biodistribution was observed in himans with rapid renal metabolism. Effective dose (ICRP 103) was 0.0139mSv/MBq. A diagnostic PET scan had an effective dose of 2.21mSv (without CT). The greatest absorbed organ dose was found in the urinary bladder (0.064mSv/MBq) followed by the kidneys (0.0225mSv/MBq), spleen (0.0081mSv/MBq), heart (0.0069mSv/MBq), liver (0.0054mSv/MBq) and lymph nodes (0.0044mSv/MBq).
In conclusion, we set-up an efficient and reproducible kit to radiolabel IL2 in GMP conditions, with 68Ga at room temperature. Results in mice and humans showed excellent biodistribution with favourable dosimetry. Clinical trials are needed to optimize its role in immune-mediated diseases and cancer.
Author
Sapienza University of Rome