Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide☆
Introduction
Selected infectious and inflammatory foci can be visualized accurately with radiolabelled cytokines, which act through interaction with specific cell-surface receptors expressed on known cell populations [1], [2]. During the past few years, several authors have described the use of cytokines, such as IL-1, which showed specific uptake at the side of infection caused by focal Staphylococcus aureus accumulation [3], [4]; IL-6 for targeting of acute inflammation [5]; IL-8 for imaging infectious foci, sterile inflammation and osteomyelitis in an animal model and in humans [6], [7], [8]; and IL-2 for targeting T lymphocytes and monocytes in chronic, mononuclear cell-mediated inflammatory processes such as autoimmune diseases [9], [10], [11], [12], [13], kidney graft rejection [14] and lymphocitic infiltration in melanoma characterized by overexpression of CD25 [15].
Chronic inflammation has been successfully targeted by radiolabelled IL-2 by means of specific binding to IL-2 receptors, expressed on activated lymphocytes. It was previously shown by Signore et al. [16] that lymphocytic infiltration in the pancreas could be visualized with 123I-labelled IL-2 within 1 h after injection. Studies in patients with autoimmune disorders such as Hashimoto thyroiditis, Graves' disease, Crohn's disease and celiac disease demonstrated localization of 123I- or 99mTc-labelled IL-2 at the side of lymphocytic infiltration [17], [18], [19], [20], [21].
Therefore, radiolabelled IL-2 is the best available agent for in vivo targeting of mononuclear cell infiltration as present in autoimmune diseases. However, the availability of 123I- or 99mTc-labelled IL-2 is limited and their preparation is laborious. For clinical application, a simple and rapid labelling procedure of recombinant human interleukin-2 (rhIL-2), using technetium-99m, is preferable. Chianelli et al. [20] reported a two-step synthesis of 99mTc-IL-2 using the bifunctional chelating agent S-tetrahydrofurfurylacetyl-(thio-2,3,5,6-tetrafluorophenyl)-adipylglycylglycine, a ligand with two active sites: one providing a N,S set of donor atoms for coordination of 99mTc, and the other, a tetrafluorophenyl-active ester, for protein conjugation via the amino groups on lysine residues of IL-2.
An alternative to this method, for 99mTc-labelling of rhIL-2 using hydrazinonicotinamide (HYNIC) [22], [23] as a bifunctional chelator, was recently reported. The HYNIC group is of particular interest because it can be easily labelled with high efficiency (rapid and high-yield radiolabelling). The possibility of developing a dry kit formulation for a convenient preparation of 99mTc-HYNIC–rhIL-2 was therefore examined.
Section snippets
Chemicals
Recombinant human IL-2 (Proleukin) was purchased from Chiron Corporation. It is a highly purified protein with a molecular weight of approximately 15,300 Da. The recombinant form differs from native IL-2 in the following manner: rhIL-2 is not glycosylated, it does not have N-terminal alanine, it has serine substituted for cysteine at amino acid position 125 and the aggregation state of rhIL-2 is likely to be different from that of native IL-2. One vial of Proleukin contains 18 million IU (1.2
Conjugation of rhIL-2 and HYNIC
The molar excess of HYNIC in the reaction mixture influenced the number of HYNIC groups introduced into the rhIL-2 molecule (Table 1). The increase of HYNIC to rhIL-2 molar ratio resulted in a simultaneous increase in the percentage of aggregates in the conjugates (determined by GF-HPLC, UV 280 nm).
Effect of different HYNIC/rhIL-2 molar ratios on 99mTc labelling
It was observed that increasing the conjugation ratio of HYNIC to rhIL-2 resulted in the rise of radiolabelling yields of the conjugates (Fig. 1). However, an increase of radiolabelled aggregate
Conclusions
Our study shows that the selection of the substitution ratio of HYNIC molecules and the peptide content in the final preparation is based on the compromise between the radiochemical yield, specific activity of the tracers, as well as on their stability and biological properties. The HYNIC–rhIL-2 conjugates with HYNIC substitution ratio from ca. 2 to ca. 4, in amounts of 10–30 μg per vial and applications of the two co-ligands (tricine and NA), ensure an efficient preparation of 99m
References (33)
- et al.
Accumulation of 125iodine labeled interleukin-2 in the pancreas of NOD mice
J Autoimmun
(2001) - et al.
Analysis of activated T cell infiltrated in rat renal allografts by gamma camera imaging after injection of 123-iodine-interleukin 2
Transpl Immunol
(1993) - et al.
The development of technetium-99m labelled interleukin-2: a new radiopharmaceutical for the in vivo detection of mononuclear cell infiltrates in immune-mediated diseases
Nucl Med Biol
(1997) - et al.
Labeling proteins with Tc-99m via hydrazinonicotinamide (HYNIC): optimization of the conjugation reaction
Nucl Med Biol
(2000) - et al.
Release of recombinant human interleukin-2 from dextran-based hydrogels
J Controlled Release
(2002) - et al.
Plasma protein binding of 99mTc-labeled hydrazino nicotinamide derivatized polypeptides and peptides
Nucl Med Biol
(2001) - et al.
Imaging of infection in rabbits with radioiodinated interleukin-1 (alpha and beta), its receptor antagonist and a chemotactic peptide: a comparative study
Eur J Nucl Med
(1998) - et al.
Radiolabelled cytokines for imaging chronic inflammation
Braz Arch Biol Technol
(2002) - et al.
Binding and internalization of interleukin-1 by T-cells
J Exp Med
(1986) - et al.
Specific targeting of infectious foci with radioiodinated human recombinant interleukin-1 in an experimental model
Eur J Nucl Med
(1995)
Targeting inflammation with radiolabeled interleukin-1 and other cytokines in various mouse models
Nucl Med Commun
Specific and rapid scintigraphic detection of infection with 99mTc-labeled interleukin-8
J Nucl Med
Radiolabeled interleukin-8 specific scintigraphic detection of infection within a few hours
J Nucl Med
99mTc-Interleukin-8 for imaging acute osteomyelitis
J Nucl Med
123I-Interleukin-2 scintigraphy: a new approach to assess disease activity in autoimmunity
J Pediatr Endocrinol Metab
Imaging active lymphocytic infiltration in coeliatic disease with iodine-123-interleukin-2 ant its response to diet
Eur J Nucl Med
Cited by (0)
- ☆
This work has been supported in part by grants [no. 2 P05A 024 28 (2005–2008) and no. 2 P05B 003 28 (2005–2008)] provided by the Ministry of Scientific Research and Information Technology, Poland.