Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide

Introduction: Interleukin-2 (IL-2) when radiolabelled with (99m)Tc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of (99m)Tc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation.

Methods: Various molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The (99m)Tc-labelling was optimized using various amounts of HYNIC-rhIL-2, (99m)Tc, SnCl(2), tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats.

Results: Generally, the highest radiolabelling yields were achieved when the HYNIC-rhIL-2 conjugates of ca. 2-4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of (99m)Tc-HYNIC-rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the (99m)Tc-HYNIC-rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 μg HYNIC-rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl(2). The monomer of (99m)Tc-HYNIC-rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed.

Conclusions: Our study shows that rhIL-2 can be efficiently radiolabelled with (99m)Tc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use (99m)Tc-HYNIC(tricine,NA)-rhIL-2 within 1 h.