Development of methods for 99mTc labelling of interleukin-2

Interleukin-2 (IL-2), a 15.4 kDa protein containing 134 amino acids is a naturally occurring cytokine which has been labelled and used as radiopharmaceutical to image inflammation and infection. The goal of our work was to develop the method of 99mTc-labelling of IL-2 using a commercially available Proleukin. The IL-2 was conjugated with HYNIC (1:5 molar ratio) and purified on Sephadex G-25 Fine. A conjugate was stored in 0.1M PB with 0.1% SDS and 0.02% NaN3. HYNIC-IL2 was labelled under reducing conditions (5-25 mg of SnC12) in the presence of Tricine and EDTA. The influence of pH was tested in the range 2 to 9.5. Radiochemical purity, RCP, was measured by HPLC, BioSEP SEC-4000 column (Phenomenex) and 0.1M PB as a solvent at 280 nm and with on-line radiometric detector and by TLC using ITLC-SG strips and 0.9% NaCl for free 99mTcO4- (Rf=l), and 0.1M citrate buffer pH=5.5 for assessment of 99mTc-colloids (Rf=0) while free 99mTcO4- and 99mTc-HYNIC-IL2 migrate with the solvent front. Stability of 99mTc-HYNIC-IL-2 in human serum was estimated by incubation of 100 ml of human serum with 20 ml of 99mTc-HYNIC-IL-2 at 37 degree C for 0.5, 1, 2, 4, 6 12 and 24 hr and analyzed by HPLC and TLC. RCP of 99mTc-HYNIC-IL2 with about 20 molar excess of Tricine varied in the range from 50% to 90%. Further optimization of the method is planned. The work has been done within the EU program COST B12. (authors)