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

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Abstract

Introduction

Interleukin-2 (IL-2) when radiolabelled with 99mTc 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 99mTc-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 99mTc-labelling was optimized using various amounts of HYNIC–rhIL-2, 99mTc, SnCl2, 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 99mTc-HYNIC–rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the 99mTc-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 SnCl2. The monomer of 99mTc-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 99mTc 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 99mTc-HYNIC(tricine,NA)-rhIL-2 within 1 h.

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)

  • Van der LakenC.J. et al.

    Targeting inflammation with radiolabeled interleukin-1 and other cytokines in various mouse models

    Nucl Med Commun

    (1997)
  • RennenH.J. et al.

    Specific and rapid scintigraphic detection of infection with 99mTc-labeled interleukin-8

    J Nucl Med

    (2001)
  • Van der LakenC.J. et al.

    Radiolabeled interleukin-8 specific scintigraphic detection of infection within a few hours

    J Nucl Med

    (2000)
  • GratzS. et al.

    99mTc-Interleukin-8 for imaging acute osteomyelitis

    J Nucl Med

    (2001)
  • SignoreA. et al.

    123I-Interleukin-2 scintigraphy: a new approach to assess disease activity in autoimmunity

    J Pediatr Endocrinol Metab

    (1996)
  • SignoreA. et al.

    Imaging active lymphocytic infiltration in coeliatic disease with iodine-123-interleukin-2 ant its response to diet

    Eur J Nucl Med

    (2000)
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    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.

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