Radiolabelling of rh-interleukin-2 with 99mc using HYNIC-(tricine)2 and evaluation of its biological activity in vitro

Full text: Introduction: Aim of the study was the optimisation of 99mTc-labelling of rhIL2 by using HYNIC and tricine as co-ligand in a pre-labelling approach and the evaluation of the biological activity of the new conjugate. Materials and Methods: HYNIC (10 mg/ml) was conjugated to 200 g of rhIL2 (2.4 mg/ml) at different pH (7.2, 8.5 and 9.5) and different HYNIC/IL2 ratios (3:1, 6:1, 12:1) for 2 hrs/darkness and at room temperature. The HYNIC-IL2 complex was purified by tC2 Sep-Pak Cartridge using a step-gradient of H2O/ethanol acidified with 25% phosphoric acid. Radiolabelling of the HYNIC-IL2 complex was performed with 99mTcO4 (5-10mCi) at different pH (4, 5 and 7), at room temperature, using tricine as co-ligand (12 μl at 100 mg/ml) and SnCl2 dihydrate(2 mg/ml) in 0.1 mM HCl.LE of 99mTc-HYNIC-IL2 was assessed by ITLC-SG using saline and acetone. Colloids were measured on BSA (5mg/ml) absorbed ITLC strips using Et-OH/NH3/H2O (2:1:5) as mobile phase. SDS-PAGE electrophoresis was performed to assess the molecular structure of 99mTc-HYNIC-IL2. In vitro stability studies of 99mTc-HYNIC(tricine)2-IL2 were evaluated by ITLC-SG after 1, 3, 6 and 20 hrs at 370C in saline, human plasma. The biological activity of the IL2 conjugated with HYNIC-NHS has been assessed in a MTT assay by incubation of CTLL-2 cells, an IL2-dependent cytotoxic T-cell line, with increasing concentration of the conjugate from 0,1 to 100 UI/ml. Following, the absorbance has been measured with a microplate reader at a test wavelength of 570 nm to a reference wavelength of 620 nm (to read the MTT formazan crystals and the unreduced MTT, respectively). Results: A stable HYNIC/IL2 conjugate was obtained at pH 8.5 using a ratio 12:1 (HYNIC:IL2). No modification of the structure of IL2 was found during the conjugation, the purification and the labelling procedure assessed by SDS-PAGE autoradiography. 99mTc-HYNIC(tricine)2-IL2 was labelled to a labelling efficiency 90% with a radiochemical purity 95% after purification, and a specific activity 200 Ci/g. The stability was always 90% in saline, plasma and in DTPA at all time points. The MTT assay study showed that the IL2 conjugated to HYNIC-NHS maintains its biological activity and that the proliferation of the CTLLs is proportional to the increasing concentration of the HYNICIL2 (from 0 to 100 UI/ml of the complex) as well as fresh unlabelled Il2. Conclusion: Using this labelling approach we obtained 99mTc- HYNIC(tricine)2-IL2 with high LE and stability. In the biological assays using the MTT salt, as indicator of the proliferation, the HYNIC-IL2 maintains unaffected the ability to induce cell proliferation. These results indicate the HYNIC-IL2 is a promising radiopharmaceutical to be formulated as 1 step kit for clinical routine use. (author)