Nuclear Medicine & Biology, Vol. 24, pp. 579-586, 1997 Copynght 0 1997 Elsevier Science Inc. ELSEVIER ISSN 0969-8051/97/$17.00 + 0.00 PII SO969-8051(97)00021-8 The Development of Technetium-99mHLabelled Interleukin-2: A New Radiopharmaceutical for the In Viva Detection of Mononuclear Cell Infiltrates in ImmuneHMediated Diseases Marco Chianelli, 1 ,2 Albert0 Signore,2 Alan R. Fritsber$ and Stephen J. Mather’” ‘NUCLEAR MEDICINE RESEARCH LABORATORY, ST. BARTHOLOMEW’S HOSPITAL, LONDON EClA 7BE UK; ‘NUMED GROUP, SERVIZIO DI MEDICINA NUCLEARE, 11CLINICA MEDICA. UNIVERSITY OF ROME “LA SAPIENZE,” ITALY; ‘NEORX CORPORATION, SEATTLE, WA ABSTRACT. We describe here a new method for labelling interleukin-2 (IL-2) in high specific activity with 99mTc for in viva studies in man. Labelling was performed via a two-step reaction using an N,S bifunctional chelating agent. To optimise the reaction, factors affecting the incorporation of 99mTc into the N,S ligand were studied. The conjugation of the preformed N,S chelate ligand to IL-2 was then similarly optimised. Various strategies for purifying the 99”Tc-IL-2 were explored including size-exclusion, ion-exchange, and several modes of reversed-phase chromatography. The radiochemical purity of the purified protein was determined by HPLC, ITLC, TCA precipitation, and SDS-PAGE. Th e receptor binding capacity of 99mTc-IL-2 was studied. Biodistribution studies in normal mice were performed with 99mTc-IL-2 purified using different techniques or labelled after prolonged storage and compared to ‘251-IL-2. NUCL MED BIOL 24;6: 579-586, 1997. 0 1997 Elsevier Science Inc. KEY WORDS. Technetium-99m, Interleukin-2, Immune-mediated diseases, Bifunctional chelating agents, Radiolabelling INTRODUCTION We have recently described the use of radioiodinated interleukin-2 (IL-2) for the early diagnosis of chronic lymphocytic infiltration. Mechanistic studies in animal models have shown that IL-2 labelled with iodine-123 ( 1231) specifically binds to infiltrating lymphocytes in viwo (21, 22), and in recent studies in man, lz31-lL-2 has proven to be of use in the detection of immune-mediated conditions such as Type 1 diabetes, coeliac disease, and Hashimoto thyroiditis (20). Because lz31 is a cyclotron-produced radionuclide, it is very expen- sive and has only limited availability. The ready availability and low cost of technetium-99m (99mTc) suggest that this would be the isotope of choice for such applications. Several methods have been described for labelling proteins and peptides with 99mTc (4, 9, 16). These were, however, primarily developed for large proteins such as albumin or monoclonal anti- bodies; interleukin-2, by contrast, is a small, relatively fragile protein, and it is essential to retain its receptor binding capacity if it is to fulfill its targeting potential in uivo (11). Moreover, IL-2 is a biologically active cytokine, and tracer amounts must be adminis- tered to patients for diagnostic studies to avoid the occurrence of pharmacological effects (5, 15). We have, therefore, undertaken a programme to develop a method for labelling interleukin-2 with 99mTc and to apply this new radiopharmaceutical in man, for the in wivo diagnosis of lymphocytic infiltration. In this paper we describe the high specific activity 99mTc labelling of IL-2, its in vitro characterisation and receptor binding, and the development of a simple purification method for routine clinical studies. The effects of aging and of different *Author for correspondence; E-mail: mather@europa.lif.icnet.uk Received 21 September 1996. Accepted 24 February 1997. purification methods on IL-2 structure and biodistribution in normal animals were also investigated. MATERIALS AND METHODS Ligund ihelling For the gentle labelling of IL-2 with 99mTc we used a preformed chelate approach based on the use of a bifunctional chelating agent, S-tetrahydrofurfurylacetyl (thio-2,3,5,6-tetrafluorophenyl)-adipyl glycylglycine, a ligand with two active sites: one providing an N,S set of donor atoms for coordination of 99mTc and the other, a tetrafluorophenyl-active ester, for protein binding via the amino groups on lysine residues (Fig. 1) (7, 12, 24). The thiol group of the chelator is protected by a tetrahydropyran group. The ligand is first complexed with 99mTc and then conjugated to the protein. Complexation of the N,S ligand was performed via transchela- tion from a labile complex with gluconic or glucuronic acid after reduction of 99mTc at acid pH with stannous chloride in the presence of isopropyl alcohol (IPA) and gentisic acid. Heating, in the presence of 99mT~, was necessary for the removal of the protective group and complexation of 99mTc. Gentisic acid was used as an antioxidant and IPA to aid solubilisation of the ligand. All reagents were freshly prepared for each experiment and were dissolved in water for injection deoxygenated under a stream of nitrogen for 30 min. Stannous chloride (anhydrous) was dissolved in 5% aqueous sulfuric acid and the ligand in 2% acetic acid in isopropyl alcohol. All reagents and parameters influencing the reaction were stud- ied to obtain the maximum ligand specific activity and labelling efficiency. Sodium [99mTc] pertechnetate 99mT~04- (l-20 GBq/ mL, 27-540 mCi/mL) and the ligand (16-82 bg/mL) were incu- bated (7-25 min) at 75-lOO”C, with different concentrations of isopropyl alcohol (9-36%), glucuronic acid (2-16 p,g/mL) or