RADIOPHARMACEUTICAL COMPOUND AND COMPOSITION FOR POSITRON EMISSION TOMOGRAPHY (PET) IMAGING OF INTERLEUKIN-2 RECEPTOR POSITIVE CELLS, PROCESS FOR THE PREPARATION THEREOF, RELATED KIT AND USES THEREOF

Abstract

The present invention relates to a radiopharmaceutical compound or composition for Positron Emission Tomography (PET) imaging of interleukin-2 (IL2) receptor positive cells, in particular, .sup.68Ga-radiolabelled interleukin-2 such as a desalanyl-1, serine-125 human interleukin-2 (dsIL2) radiolabelled with a short-lived PET radioisotope (or radionuclide) gallium-68 (.sup.68Ga) by using tris-(hydroxypyridinone-maleimide) (THP-mal) as a chelator. The invention concerns also a kit comprising dsIL2 linked to THP-mal which can be added with .sup.68Ga in order to obtain the above mentioned radiopharmaceutical, at room temperature, suitable for PET imaging, a process for the preparation of the radiopharmaceutical and its use in medical and diagnostic field.

Claims

1. Radiopharmaceutical compound for the imaging or for locating interleukin-2 receptor positive cells, such as activated T lymphocytes, said compound comprising or consisting of a protein labelled with a radioisotope by a chelator, wherein said protein is a protein consisting of recombinant human desalanyl-1, serine-125 human interleukin-2, a fragment or a mutant thereof having at least one Cysteine residue, which is capable to bind interleukin-2 receptors alpha and/or beta; said radioisotope is a positron-emitting radioisotope, such as .sup.68Ga and .sup.64Cu; and said chelator is a chelator with a reactive group able to bind a SH group of said recombinant human desalanyl-1, serine-125 human interleukin-2, fragment or mutant thereof and to chelate the radioisotope, at room temperature.

2. Radiopharmaceutical compound according to claim 1, wherein said chelator is a maleimide-coupled metal chelator such as tris(hydroxypyridinone)-maleimide,1,4,7-triaza-cyclononane (THP-mal) having formula C44H57N9O13 or maleimide-1-glutaric acid-4,7-acetic acid (NODAGA-mal).

3. Radiopharmaceutical compound according to claim 1, wherein said protein is recombinant human desalanyl-1, serine-125 human interleukin-2 and said chelator is THP-mal.

4. Precursor compound of the radiopharmaceutical compound as defined by claim 1, said precursor comprising or consisting of a protein conjugated with a chelator, wherein said protein is a protein consisting of recombinant human desalanyl-1, serine-125 human interleukin-2, a fragment or a mutant thereof having at least one Cysteine residue, which is capable to bind interleukin-2 receptors alpha and/or beta; and said chelator is a chelator with a reactive group able to bind a SH group of said recombinant human desalanyl-1, serine-125 human interleukin-2, fragment or mutant thereof and to chelate a positron-emitting radioisotope, such as .sup.68Ga and .sup.64Cu, at room temperature.

5. Precursor compound according to claim 4, wherein said chelator is a maleimide-coupled metal chelator such as tris(hydroxypyridinone)-maleimide,1,4,7-triaza-cyclononane (THP-mal) having formula C44H57N9O13 or maleimide-1-glutaric acid-4,7-acetic acid (NODAGA-mal).

6. Precursor compound according to claim 4, wherein said protein is recombinant human desalanyl-1, serine-125 human interleukin-2 and said chelator is THP-mal.

7. Radiopharmaceutical composition comprising or consisting of the radiopharmaceutical compound as defined in claim 1, in association with one or more excipients and/or adjuvants.

8. Pharmaceutical composition comprising or consisting of the precursor compound as defined in claim 4, in association with one or more excipients and/or adjuvants.

9.-14. (canceled)

15. A method of in vivo evaluating T cell infiltration in active inflammatory and infective disease in a subject, the method comprising administering to the subject a radiopharmaceutical compound according to claim 1.

16. A method of in vivo locating or imaging interleukin-2 receptor positive cells in a subject, the method comprising administering to the subject a radiopharmaceutical compound according to claim 1.

17. An imaging agent for PET imaging comprising the radiopharmaceutical compound according to claim 1.

18. Method for obtaining a radiopharmaceutical compound according to claim 1, said method comprising: a) conjugating a protein with a chelator in order to obtain a precursor compound of the radiopharmaceutical compound, wherein said protein is a protein consisting of recombinant human desalanyl-1, serine-125 human interleukin-2, a fragment or a mutant thereof having at least one Cysteine residue, which is capable to bind interleukin-2 receptors alpha and/or beta; and said chelator is a chelator with a reactive group able to bind a SH group of said recombinant human desalanyl-1, serine-125 human interleukin-2, fragment or mutant thereof and to chelate a positron-emitting radioisotope, such as .sup.68Ga and .sup.64Cu, at room temperature.

19. Method according to claim 18, further comprising: b) radiolabelling with a positron-emitting radioisotope, such as .sup.68Ga and .sup.64Cu, the precursor compound obtained in step a), at room temperature.

20.-21. (canceled)

22. Kit for the preparation of a radiopharmaceutical compound according to claim 1, said kit comprising or consisting of: a first vial comprising a precursor compound comprising or consisting of a protein conjugated with a chelator, wherein said protein is a protein consisting of recombinant human desalanyl-1, serine-125 human interleukin-2, a fragment or a mutant thereof having at least one Cysteine residue, which is capable to bind interleukin-2 receptors alpha and/or beta; and said chelator is a chelator with a reactive group able to bind a SH group of said recombinant human desalanyl-1, serine-125 human interleukin-2, fragment or mutant thereof and to chelate a positron-emitting radioisotope, such as .sup.68Ga and .sup.64Cu, at room temperature, or a pharmaceutical composition comprising or consisting of the precursor compound in association with one or more excipients and/or adjuvants.

23.-24. (canceled)

25. Method according to claim 15, comprising PET imaging.

26. Method according to claim 16, comprising PET imaging.

27. A method of in vivo evaluating T cell infiltration in active inflammatory and infective disease in a subject, the method comprising administering to the subject a radiopharmaceutical composition according to claim 7.

28. A method of in vivo locating or imaging interleukin-2 receptor positive cells in a subject, the method comprising administering to the subject a radiopharmaceutical composition according to claim 7.

29. Method according to claim 27, comprising PET imaging.

30. Method according to claim 28, comprising PET imaging.

Description

[0064] The present invention is described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:

[0065] FIG. 1 shows ESI characterization of unconjugated dsIL2 (*) and THP-mal conjugated dsIL2 (**). This graph proves that the difference in the mass is caused by the addition of a THP-mal group after the conjugation step.

[0066] FIG. 2 shows the results of the saturation binding assay of radiolabeled IL2 on activated T cells; the graph shows total binding (circles), unspecific binding (squares) and specific binding (triangles) to IL2 receptor.

[0067] FIG. 3 shows the results of the Immunoreactive fraction assay of radiolabeled IL2 on activated T-cells; The graph is an inverted plot from which the formula to calculate the % IRF has been extrapolated.

[0068] FIG. 4 shows the results of the biodistribution of radiolabeled IL2 in normal BALB/C mice; The graph shows the percentage of injected dose per gram (% ID/g) in major organs after 15 (white), 60 (grey) and 120 (black) minutes from injection.

EXAMPLE 1: METHOD OF RADIOLABELLING DSIL2 WITH .SUP.68.GA AND PREPARATION OF A LYOPHILIZED KIT

[0069] Experiments Performed to Set-Up a Kit for Radiolabelling Interleukin-2 with .sup.68Ga

[0070] Several experiments have been set up to find the best concentrations of dsIL2, THP-mal and buffers to obtain the best conjugation and labelling conditions.

[0071] THP-mal:dsIL2 ratios of 5:1, 10:1, 20:1, 40:1 (i.e. from 5:1 to 40:1) have been tested and it was found that for all of them the chelating agent binds to the dsIL2 molecule, generating a single species of precursor, as revealed by liquid chromatography-Mass Spectroscopy analysis (ESI) shown in FIG. 1. This is a very relevant finding, since, this allow us to prevent the formation of different conjugates containing more than one THP-mal group, thus potentially reducing the affinity of the radiopharmaceutical for its receptor. Therefore, it is possible to produce only one species, and not a mixture of THP-mal-dsIL2 conjugates with different structures. The kit, which contains THP-mal-dsIL2, can be reconstituted with 0.5-1 ml of generator eluate containing up to 300 MBq of .sup.68Ga in HCl 0.1 M, at room temperature, gently mixing for 10 minutes, with or without using a syntheses module. The content of the kit is sterile and sterility will be preserved in each passage and can be directly injected into the patient. The amount of .sup.68Ga-THP-mal-dsIL2 obtained with 300 MBq of .sup.68Ga can be used for 1 or 2 patients within 1 h from the syntheses.

[0072] Preparation of a Kit for Radiolabelling Interleukin-2 with .sup.68Ga

[0073] A lyophilized kit composed of 2 vials and an adapter has been prepared using:

[0074] a vial 1 containing 160 μg of lyophilized THP-mal-dsIL2;

[0075] a vial 2 containing 300 μl of an ammonium acetate solution in H2O (pH=5); and optionally

[0076] a luer-lock compatible adapter with which vial 1 can be directly placed on a pre-made cassette of any automated synthesizer of radiopharmaceuticals.

[0077] For the preparation of vial 1, THP-mal (a THP derivative, tris(hydroxypyridinone)-maleimide, Chemical Formula C44H57N9O13) in a 20:1 ratio is added dropwise, over 10 minutes with gentle mixing, to a solution containing 160 μg of dsIL2 in mannitol and sodium dodecyl sulphate, buffered with monobasic and dibasic sodium phosphate to a pH of 7.5 (range 7.2 to 7.8). After 1 h incubation at room temperature, the product is purified by size exclusion chromatography using 0.9% NaCl as eluent and the conjugate is filtered through a 0.22 μm filter and then lyophilized under nitrogen atmosphere in a 10 ml crimped, glass vial compatible with any automated synthesizer of radiopharmaceuticals that does not use pre-made cassettes.

[0078] For the preparation of vial 2, 300 μl of an ammonium acetate solution 0.1 M (pH=5) are filtered through a 0.22 μm filter and placed in a 1 ml crimped glass vial under nitrogen atmosphere.

[0079] For .sup.68Ga labelling, the content of vial 2 is transferred to vial 1. After gently mixing, 0.5-1 ml of .sup.68Ga eluate in HCl 0.1 N (100-300 MBq, in this case approximately 300 MBq, of freshly eluted .sup.68Ga in HCl 0.1 M) are added to vial 1 containing 160 μg of THP-dsIL2, and ammonium acetate (0.1 M, pH=5). After 10 minutes incubation at room temperature, the solution in vial 1 is ready to be injected into the patient. To use the kit with an automated synthesizer, it is possible to transfer the content of vial 2 into vial 1 and then, through the luer-lock adapter, it is possible to mount the vial in a virgin cassette to directly elute .sup.68Ga from the generator into vial 1. Alternatively, this can be performed by eluting the generator with a peristaltic pump directly into the vial containing the precursor.

[0080] This procedure will produce .sup.68Ga-THP-mal-dsIL2 with retention of its biological activity and receptor binding activity.

[0081] At the end of the incubation, the solution is filtered with a 0.22 μm filter (Millex GV, Millipore) and quality controls can be performed.

[0082] Quality Controls

[0083] Quality controls are performed by both reverse phase HPLC (RP-HPLC) and size exclusion HPLC.

[0084] RP-HPLC can be performed using a kinetex C18 column (Phenomenex) and a gradient of H.sub.2O and ACN as mobile phases (0-5 min 5% ACN; 5-15 50% ACN; 15-25 95% ACN; 25-35 5% ACN). Size exclusion HPLC can be performed using a Yarra column (Phenomenex) and 0.1 M phosphate buffer as mobile phase (isocratic).

[0085] Stability of the radiopharmaceutical has been tested against 0.9% NaCl solution and human serum donated by normal volunteers after signing written consent up to 3 h.

[0086] Radiochemical purity is determined using ITLC-SG strips developed with 0.1M HCl to quantify levels of ionic .sup.68Ga, and ITLC-SG strips developed with 1:1 MeOH:1 M NH4OAc to quantify colloidal .sup.68Ga-hydroxide plus ionic .sup.68Ga. The strips can be analyzed with a Bioscan AR-2000 radiochromatogram scanner fitted with high-resolution collimator. Negligible amount of free or colloidal .sup.68Ga should be observed (<5%).

[0087] Saturation Binding Assay

[0088] In order to verify the receptor binding capacity of radiolabelled IL2, T-cells were isolated from peripheral blood mononuclear cells (PBMNCs) from healthy donors (after signing written consent) by centrifugation on a standard Ficoll/Hyplaque density gradient. Cells were cultured for 48 to 72 h at 106/mL in complete culture medium and 1 kg/mL purified phytohemagglutinin (PHA) (Mirux) at 37° C. Before the assays, cells were incubated for 60 min at 37° C. in RPMI medium to remove endogenous IL2 from the cell-surface IL2R and then washed twice and resuspended in iced 1% BSA phosphate-buffered saline (PBS) containing 0.01% sodium azide (4° C.).

[0089] Then, 3×10.sup.6 cells were placed in triplicate in Eppendorf vials and incubated with decreasing concentrations of .sup.68Ga-THP-mal-dsIL2 for 1 hour at 4° C., to calculate total binding curve. The same experiment was performed in the presence of a 100 fold molar excess of unlabelled dsIL2 to each vial, to calculate non-specific binding. At the end of the incubation time, the cells were washed twice with 0.5 ml of PBS. After centrifugation, cell pellets and the supernatants were counted separately in a single-well gamma counter (Perkin Elmer). Data were analyzed using Prism Graphpad software, as shown in FIG. 2, and revealed a Kd value of 1.79 nM.

[0090] Immunoreactive Fraction (IRF) Assay

[0091] IRF assay was performed as described by Lindmo et al. Briefly, cells were seeded in Eppendorf vials (from 8×106/mL to 0.4×106/mL) and in each vial .sup.68Ga-THP-mal-dsIL2 was added at constant concentration (10 nM). After 1 h incubation at 4° C., the vials were centrifuged at 13000 rpm (5000 g) for 3 minutes and the supernatant was collected. This step was repeated after washing the pellet with 0.5 ml of PBS.

[0092] Radioactivity associated with pellets and supernatants was then determined by counting each vial with a single-well gamma counter (Perkin Elmer). Data were analyzed using Prism Graphpad software and an IRF of 78.4% was obtained, as shown on FIG. 3, demonstrating that the majority of dsIL2 is radiolabelled and capable to bind to its receptor.

[0093] Biodistribution in Normal BALB/C Mice

[0094] Biodistribution studies were performed in 12 normal BALB/C mice. Each animal received an intravenous injection (tail vein) of 0.55 MBq (in 1000 μl) of .sup.68Ga-THP-mal-dsIL2 according to the present invention. After 15, 60 and 120 minutes from the injection, four animals per time point were sacrificed to collect major organs and blood (collected samples included intestine, kidneys, spleen, stomach, liver, muscle, bone, lungs, heart and salivary glands). Each sample was weighted and counted with a single-well gamma counter to determine radioactivity. Data were expressed as percentage of injected dose per gram of tissue (% ID/g). FIG. 4 shows the results of the biodistribution that highlight a higher % ID/g in kidneys, where native human IL2 is normally metabolized and lower % ID/g in the liver, if compared with other radiolabeled IL2 species. This strengthen the hypothesis that binding of the THP-mal chelating agent to SH groups instead of the NH2 at the N-terminus, confers to the radiolabeled IL2 a more favorable biodistribution.