Cellular Targeted Label Delivery System

Abstract

The present invention relates to an isolated cellular targeted delivery system comprising a CD45+ leukocyte cell comprising within said cell a complex of one or more iron binding proteins and/or a label as well as methods for producing such isolated cellular targeted delivery system and uses of such system for therapy diagnosis and in particular for diagnosis of cancer, particularly metastatic cancer, in particular for therapy of cancer.

Claims

1-24. (canceled)

25. A method for diagnosing a tumor, comprising administering to a subject in need thereof, a CD45+ leukocyte cell comprising within said cell a complex of one or more iron binding proteins and a label to diagnose the tumor.

26. The method of claim 25, wherein the label is encapsulated by the one or more iron binding protein or multimers thereof.

27. The method of claim 26, wherein there is no covalent or non-covalent bond between the one or more iron binding protein and the label.

28. The method of claim 27, wherein the one or more iron binding protein is ferritin.

29. The method of claim 25, wherein the CD45+ leukocyte cell is producible from a CD34+ hematopoietic precursor cell.

30. The method of claim 25, wherein the CD45+ leukocyte is selected from the group consisting of a monocyte, a differentiated monocyte, a lymphocyte and a granulocyte.

31. The method of claim 25, wherein (i) the leukocyte is a monocyte, wherein the monocyte is a CD11b.sup.+ monocyte, (ii) the leukocyte is a differentiated monocyte, wherein the differentiated monocyte is selected from the group consisting of a macrophage, an activated macrophage, or a dendritic cell, (iii) the leukocyte is a lymphocyte, wherein the lymphocyte is selected from the group consisting of a CD3.sup.+ and CD4.sup.+ or CD8.sup.+ T lymphocyte, or a CD19.sup.+, CD20.sup.+, CD21.sup.+, CD19.sup.+ CD20.sup.+, CD19.sup.+ CD21.sup.+, CD20.sup.+ CD21.sup.+, or CD19.sup.+ CD20.sup.+ CD21.sup.+ B lymphocyte, (iv) the leukocyte is a granulocyte, wherein the granulocyte is selected from the group consisting of a neutrophil, an eosinophil and a basophil.

32. The method of claim 25, wherein the CD45+ leukocyte is a differentiated monocyte, wherein the differentiated monocyte is an activated macrophage, wherein the activated macrophage: (i) is producible by in vitro incubation of a monocyte or macrophage with a factor capable of altering expression markers on macrophages; (ii) is characterized by expression of at least one of following antigens: CD64, CD86, CD16, CD32, high expression of MHCII, and/or production of iNOS and/or IL-12; (iii) is producible by in vitro incubation of a monocyte or macrophage with a factor capable of inducing the ability of the macrophage to phagocytose; (iv) is characterized by expression of at least one of following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC-motive chemokine receptor 4 (CXCR4), CD163, and/or T cell immunoglobulin-domain and mucin-domain 2 (TIM-2), and/or show low expression of MHCII; (v) has the ability to phagocytose; and/or (vi) is capable of cytokine secretion, or production of inducible nitric oxide synthetase (iNOS) (or other pro-inflammatory compounds), arginase or other immunosuppressive/anti-inflammatory compounds.

33. The method of claim 25, wherein (i) the M1 inducer is selected from the group consisting of LPS, INF-γ, GM-CSF and viral and bacterial infection; or (ii) the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, immune complex of an antigen and antibody, IgG, heat activated gamma-globulin, glucocorticosteroid, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonist, Leukocyte inhibitory factor, adenosine, helminth and fungal infection.

34. The method of claim 25, wherein the CD45+ leukocyte is a monocyte, wherein the monocyte: (i) is producible from a CD34.sup.+ hematopoietic precursor cell; (ii) is producible by in vitro incubation of monocytes with at least one inducer; (iii) is characterized by expression of at least one of the following antigens: TfR.sup.+, CD163.sup.+, TIM-2.sup.+, CD14.sup.+, CD16.sup.+, CD33.sup.+, and/or CD115.sup.+; (iv) is characterized by expression of at least one of the following antigens: TfR.sup.+, CD163.sup.+, TIM-2.sup.+, CXCR4.sup.+, CD14.sup.+, and/or CD16.sup.+; and/or (v) has the ability to phagocytose.

35. The method of claim 25, wherein: (i) the M1 inducer is selected from the group consisting of LPS, INF-γ, GM-CSF or viral or bacterial infection; (ii) the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, immune complex of an antigen and antibody, IgG, heat activated gamma-globulins, Glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonist, Leukocyte inhibitory factor, cancer-conditioned medium, cancer cells, adenosine and helminth or fungal infection.

36. The method of claim 25, wherein the CD45+ leukocyte is a lymphocyte, wherein the lymphocyte: (i) is obtainable from blood, spleen, or bone marrow or is producible from a CD34.sup.+ precursor cell; (ii) is an immunologically competent lymphocyte; (iii) expresses antigen specific T cell receptors; and/or (iv) is characterized by expression of at least one of the following antigens: (a) CD3.sup.+ and CD4.sup.+ or CD8.sup.+ or (b): CD19.sup.+, CD20.sup.+, CD21.sup.+, CD19.sup.+ CD20.sup.+, CD19.sup.+ CD21.sup.+, CD20.sup.+ CD21.sup.+, or CD19.sup.+ CD20.sup.+ CD21.sup.+ antigen.

37. The method of claim 25, wherein the CD45+ leukocyte is a granulocyte, wherein the granulocyte: (i) is obtainable from blood, spleen or bone marrow or producible from a CD34.sup.+ precursor cell; (ii) is characterized by expression of at least one of the following CD66b.sup.+ and/or CD193.sup.+; (iii) is a polymorphonuclear leukocyte characterized by the presence of granules in their cytoplasm; and/or (iii) is characterized by expression of at least one of the following: TfR.sup.+, CD163.sup.+, TIM-2.sup.+, and/or CXCR4.sup.+.

38. The method of claim 25, wherein the one or more iron binding protein is selected from the group consisting of ferritin, haemoglobin, haemoglobin-haptoglobin complex, hemopexin, transferrin, and lactoferrin.

39. The method of claim 25, wherein the label is selected from the group consisting of a fluorescent dye, a fluorescence emitting isotope, a radioisotope, a detectable polypeptide, a nucleic acid encoding a detectable polypeptide, and a contrast agent.

40. The method of claim 25, wherein the label comprises a chelating agent which forms a complex with divalent or trivalent metal cations.

41. The method of claim 40, wherein the chelating agent is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, Diethylenetriamine-N,N,N′,N′,N′″-pentaacetic acid (DTPA) and 6-Hydrazinopyridine-3-carboxylic acid (HYNIC).

42. The method of claim 39, wherein the label is a contrast agent that comprises a paramagnetic agent or ferrihydride.

43. The method of claim 39, wherein the label is a radioisotope or fluorescence emitting isotope selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, fluorescence emitting isotopes, as well as conjugates and combinations of above with proteins, peptides, small molecular inhibitors, antibodies or other compounds.

44. The method of claim 39, wherein the label is a fluorescence dye selected from the group consisting of the following classes of fluorescent dyes: Xanthens, Acridines, Oxazines, Cynines, Styryl dyes, Coumarines, Porphines, Metal-Ligand-Complexes, Fluorescent proteins, Nanocrystals, Perylenes and Phtalocyanines as well as conjugates and combinations of these classes of dyes.

45. The method of claim 39, wherein the label is a detectable polypeptide, wherein the detectable polypeptide is an autofluorescent protein or any structural variant thereof with an altered adsorption and/or emission spectrum.

46. The method of claim 25, wherein: (i) bond(s) between the iron binding protein(s) and the label comprised in the complex are covalent and/or non-covalent; and/or (ii) the label comprised in the complex is entrapped/encapsulated by the one or more iron binding proteins or multimers thereof.

47. A diagnostic composition comprising an isolated targeted delivery system comprising a CD45+ leukocyte cell comprising within said cell a complex of one or more iron binding proteins and a label and a pharmaceutically acceptable carrier and/or suitable excipient(s).

48. The diagnostic composition of claim 47, wherein the label is encapsulated by the iron binding protein or multimers thereof.

49. The diagnostic composition of claim 48, wherein there is no covalent or non-covalent bond between the iron binding protein and the label.

50. The diagnostic composition of claim 49, wherein iron binding protein is ferritin.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0144] FIG. 1: Panel (A) shows a minimal active fragment of a consensus amino acid sequence among mammalian ferritin H chains and two full length consensus sequences based on several mammalian ferritin H chains (see SEQ ID NO: 1, 2 and 7, respectively) as well as a minimal and full length amino acid sequence of mouse (SEQ ID NO: 3 and 4) and human (SEQ ID NO: 5 and 6) ferritin H chain. Panel (B) shows a a minimal active fragment of a consensus amino acid sequence among mammalian ferritin L chains and two full length consensus sequences based on several mammalian ferritin L chains (see SEQ ID NO: 8, 9 and 14, respectively) as well as a minimal and full length amino acid sequence of mouse (SEQ ID NO: 10 and 11) and human (SEQ ID NO: 12 and 13) ferritin L chain. Panel (C) shows a minimal active fragment of a consensus amino acid sequence among mammalian haemoglobin alpha chains and one full length consensus sequences based on several mammalian haemoglobin alpha chain (see SEQ ID NO: 15 and 16, respectively) as well as a minimal and full length amino acid sequence of human (SEQ ID NO: 17 and 18) haemoglobin alpha chain. Panel (D) shows a minimal active fragment of a consensus amino acid sequence among mammalian haemoglobin beta chains and a full length consensus sequences based on several mammalian haemoglobin beta chain (see SEQ ID NO: 19 and 20, respectively) as well as a minimal and full length amino acid sequence of human (SEQ ID NO: 21 and 22) haemoglobin beta chain. Panel (E) shows a N- and C-terminal minimal active fragment of a consensus amino acid sequence among mammalian transferrins (SEQ ID NO: 23 and 24) and a full length consensus sequences based on several mammalian transferrins (SEQ ID NO: 25) as well as a N- and C-terminal minimal active fragment of a human transferrin (SEQ ID NO: 26 and 27) and full length amino acid sequence of human transferrin (SEQ ID NO: 28). In the consensus sequences X indicates a position that is variable and stands for any natural amino acid. Preferably, in each case X in dependently of other X stands for the amino acid present in the human protein.

[0145] FIG. 2: Shows macrophage inside the mouse tumour mass (TRITC stained before injection, loaded with FITC-decorated ferritin).

[0146] FIG. 3: Shows confocal microscopy image of tumour tissue mouse injected with mammary cancer cells and given i.v. macrophages loaded with FITC-ferritin (asterix)—it is clearly observed, not only in macrophages but also in cancer cells, that ferritin-FITC spread within all tumour mass.

[0147] FIG. 4: Shows MRI images of mouse mammary tumour. The mouse was treated (at time point 0 h) with macrophages (i.v. injection) loaded with ferritin Fh. Then we observed increased diameter of blood vessels (arrow) filled with injected macrophages (giving significant T2-signal reduction) and afterword macrophages spread to the tissue (spot-like pattern; arrows). These changes (in the same time points) were observed in all examined mice.

[0148] FIG. 5: Shows ferritin, haemoglobin and transferrin uptake by macrophages, ferritin and haemoglobin uptake by monocytes and ferritin uptake by lymphocytes and granulocytes.

[0149] FIG. 6: Shows the stability of the ferritin storage by macrophages.

[0150] FIG. 7: Shows the picture from two-photon microscopy showing tumour from a mouse that received pre-labeled (before administration) macrophages containing Ferritin-FITC.

[0151] FIG. 8: Shows the whole body imaging of mice that intravenously received labeled macrophages, showing their accumulation in the tumour site and their distribution in other organs.

[0152] FIG. 9: Shows the migration of macrophages to hypoxic tissue, a cross-section of the tumour from a mouse that was administered intravenously with pre-labeled macrophages, tumour hypoxic areas are visualized with a hypoxia marker—pimonidazolone.

[0153] FIG. 10: Shows the signal recorded by PET from a whole-body analysis of mice with the metastatic 4T1 cancer. Mice received intravenously macrophages loaded with 18F-FDG. Signal accumulation is increased in the lungs of mice with micrometastases (confirmed by pathology examination). Mice receiving plain 18F-1-DG, or mice without 4T1 cancer had lower PET signal.

EXAMPLE SECTION

Example 1—Activation of Macrophages

[0154] Macrophages for use according to the present invention were obtained, differentiated and activated as follows. In order to activate macrophages, they are obtained firstly from bone marrow precursors (for example see paper: Weischenfeld and Porse, 2008, CSH Protoc, doi. 10.1101/pdb.prot.5080) or blood monocytes. Alternatively, they can be obtained from peritoneum. The methods of macrophage isolation, culture, differentiation and polarization/activation are well known for those skilled in the art. For example, they have been described in details by Murray et al. (Immunity, 2014, 41(1):14-20).

[0155] In this practical realization of the invention bone marrow derived macrophages were obtained from BALB/c or C57B1/6 mouse, however canine blood-monocyte-derived macrophages or commercially available macrophage cell lines (monocyte-macrophage lineage mouse cells: RAW 264.7, J744, human cells: THP-1, U937, or canine cells DH82).

[0156] Shortly, such bone marrow derived macrophages are seeded in plastic Petri dish in 5 ml medium (3 ml cells per plate): DMEM:F12+glutamine/glutamax+10% FBS+Penicillin/Streptomycin and 20% of L929 conditioned medium or M-CSF (50 ng/ml). In the next five days the medium is supplemented in growth factor and one of the activating compounds or their combinations as one cytokine cocktail.

[0157] Alternatively, macrophages have been cultured in “M1/M2 Macrophage Generation Medium” (Promocell) or equivalent commercially available or self-made medium containing all the necessary cytokines and interleukins to consider them as activated.

[0158] In order to obtain macrophages from blood monocytes, fresh blood (not older than 12 hours) is spin down using Histopaque system 1077 or equivalent and white blood cells (or alternatively, only white blood cells collected from the blood bank) in an appropriate amount of pre-warmed Monocyte Attachment Medium (or equivalent, e.g. DMEM/RPMI supplemented with M-CSF). e.g. 15 ml Medium per T-75 flask. A seeding density should be of 1-2 million/cm.sup.2 for mononuclear cells with a monocyte content of ≥25% and 1.5-3 million/cm2 for a monocyte content of <25%. Then, cells are incubated for 1-1.5 hours at 5% CO.sub.2 and 37° C. in the incubator without any further manipulation.

[0159] After cell attachment, they are washed at least twice, and then an appropriate amount of complete “M1- or M2-Macrophage Generation Medium DXF” is added to the cells (e.g. 20 ml per T-75 flask) and cells are incubated for 6 days at 37° C. and 5% CO.sub.2 without medium change. In order to activate macrophages, the whole medium should be replaced with medium supplemented by activating compound.

[0160] Activating compounds used in this invention (for bone-marrow derived cells or to activate cells from monocyte-macrophage cell lines) are as follows: IL-4 (20 ng/ml), IFN-γ (at least 20 ng/ml), LPS (at least 10 ng/ml), IL-13 (at least 20 ng/ml), IL-10 (at least 20 ng/ml), dexamethason (at least 20 μg/ml), oxLDL (at least 20 ng/ml), TNF-α (20 ng/ml), TGF-β (20 ng/ml), cortisol (150-300 ng/ml) or their combinations as one cytokine cocktail. In order to obtain unactivated macrophages, the activating compound has not been added.

[0161] Reverse of the polarization/activation of macrophages (from classically activated to alternatively activated) can be reached for example by culture of macrophages in appropriate cytokines listed above for at least 48 hrs.

Example 2—Monocyte Isolation

[0162] In order to obtain monocytes in this practical realization of the invention bone marrow derived or spleen-derived monocytes were obtained from BALB/c or C57B1/6 mouse, however canine blood monocyte or commercially available monocyte cell lines were used (monocyte-macrophage lineage mouse cells: RAW 264.7, J744, human: THP-1, U937, or canine DH82 cell line).

[0163] To obtain blood monocytes, fresh blood (not older than 12 hours) is spin down using Histopaque system 1077 or equivalent and white blood cells are seeded in an appropriate amount of pre-warmed Monocyte Attachment Medium (or equivalent, e.g. DMEM/RPMI supplemented with 20 ng/ml M-CSF), e.g. 15 ml Medium per T-75 flask. Alternatively, only white blood cells collected from the blood bank (buffy coat) may be used. A seeding density should be of 1-2 million/cm2 for mononuclear cells with a monocyte content of ≥25% and 1.5-3 million/cm2 for a monocyte content of <25%. Then, cells are incubated for 1-1.5 hours at 5% CO.sub.2 and 37° C. in the incubator without any further manipulation. After cell attachment, they are washed at least twice, and adherent cells are considered as monocytes.

[0164] In order to obtain bone-marrow derived monocytes, in this practical realization of the invention bone marrow derived macrophages were obtained from BALB/c or C57B1/6 mouse. Shortly, such bone marrow derived precursors are seeded in plastic Petri dish in 5 ml medium (3 ml cells per plate): DMEM:F12+glutamine/glutamax+10% FBS+Penicillin/Streptomycin and 20% of L929 conditioned medium or 20 ng/ml M-CSF. Two days later 5 ml of standard medium is added. Then, after two days 0.5 ml/plate L929 conditioned medium is added. Adherent cells are considered as monocytes.

[0165] In order to obtain spleen derived monocytes, in this practical realization of the invention, the spleen has been mechanically dissociated to obtain single cell suspension and passed through the 70 μm cell strainer. Cells were centrifuged and supernatant was removed. After erythrocyte lysis the monocytes were isolated using magnetic bead purification e.g. EasySep Mouse Monocyte Enrichment Kit protocol and appropriate magnet.

[0166] To obtain better effects of their protein loads and migration before use they may be pre-treated with macrophage activation stimuli: IL-4 (20 ng/ml), IFN-γ (at least 20 ng/ml), LPS (at least 10 ng/ml), IL-13 (at least 20 ng/ml), IL-10 (at least 20 ng/ml), dexamethason (at least 20 μg/ml), oxLDL (at least 20 ng/ml), TNF-α (20 ng/ml), TGF-β (20 ng/ml), cortisol (150-300 ng/ml) or their combinations as one cytokine cocktail.

Example 3—Granulocyte Isolation

[0167] To obtain granulocyte cells from blood, 9 parts of blood were diluted with 1 part of ACD buffer (containing 0.17 M d-glucose, 0.10 M citric acid, 0.11 M trisodium citrate). Blood from this step was further diluted with PBS at the 1:1 ratio and centrifuged. After removing plasma and buffy coat, remaining cells were mixed with PBS to 80% of the original volume from the first step (ACD-blood) and then diluted with cold distillated water at the ratio of 4:12. Then, 6 parts of 2.7% of NaCl solution were added and centrifuged. After removal of supernatant cells were resuspended in RPMI-1640 medium. These cell were considered as granulocytes.

Example 4—Lymphocyte Isolation

[0168] In order to obtain spleen derived lymphocytes, in this practical realization of the invention, the spleen has been mechanically dissociated to obtain single cell suspension and passed through the 70 μm cell strainer. Cell were centrifuged and supernatant was removed. After erythrocyte lysis the lymphocytes were isolated using magnetic bead purification e.g. EasySep Mouse CD4.sup.+ Enrichment Kit protocol and appropriate magnet.

Example 5—Preparation of Ferritin Complexes

[0169] In order to incorporate ferritins with the anticancer drug (e.g. classic drugs like cyclophosphamide, chlorambucil, melphalan, bendamustine, banoxantrone or hypoxia-activated prodrug like TH-302) or with the “imaging contrast agents” (e.g. ferrihydride or isotope) ferritins have to be prepared before macrophage treatment. Shortly, recombinant mouse proteins according to SEQ ID NO: 4 (FIG. 1) are obtained as follows. The expression vector pET-22b containing a synthetic gene encoding ferritin protein of SEQ ID NO: 4 was transformed into E. coli BL21 (DE3). E. coli culture was grown at 37° C. to OD600 0.6 in 1 L of Luria-Bertani broth (LB) added with ampicillin (100 mg/L). Protein expression was induced by addition of 1 mM isopropyl thio-b-D-galactoside (IPTG) and the culture was incubated overnight. Cells were harvested by centrifugation (15000 g for 15 min) and suspended in 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/mL DNase, 10 mM MgCl.sub.2 and disrupted by sonication. The lysate was centrifuged at 15000 g for 30 mM and the supernatant was treated 10 min at 50° C., centrifuged to remove denatured proteins and then at 70° C. for 10 min and centrifuged again. The supernatant was added with 30% (NH).sub.4SO.sub.4 at 4° C. stifling for 1 h and centrifuged at 15000 g for 30 min. The supernatant was added with 70% (NH).sub.4SO.sub.4 at 4° C. stirring for 1 h and centrifuged at 15000 g for 30 mM. The pellet was resuspended in 20 mM Hepes (pH 7.5), 150 mM NaCl and dialysed overnight at 4° C. against the same buffer. The protein was loaded on a HILOAD 26/600 SUPERDEX 200 gel-filtration column (GE-Healthcare) and then sterile filtered and stored at 4° C. (FIG. 9) Protein concentration was determined spectrophotometrically at 280 nm using a molar extinction coefficient of 21000 M.sup.−1 m.sup.−1 and by Bradford assay measuring the absorbance at 595 nm.

[0170] Said ferritins include recombinant mammalian ferritin proteins H and/or L homopolymers.

[0171] Ferritins, obtained as previously described, are purified by standard methods in order to obtain an endotoxin free, pre-clinical grade product (see, for example: Ceci et al. 2011, Extremophiles 15(3):431-439; Vanucci et al. 2012, Int J Nanomed 7:1489-1509). Shortly, the ferritin conserved sterile in a storage solution containing 20 mM Hepes pH 7.5 is diluted to a final concentration of 4 uM in 24-mer in acidic solution (final pH<3.0) or, alternatively, at highly basic pH values (pH>9.5) (see for example Pontillo et al., 2016), thus allowing the dissociation of multimer. Drugs are dissolved at very high concentrations in the appropriate solvent and then a small volume is added to the ferritin solution with a 200 molar excess. PH is then brought to neutrality by addition of appropriate amounts of NaOH/HCl solutions in order to allow multimer reconstitution. Current experimental methods indicate that three/four washings using PBS (concentration steps) in 100 kDa cut off concentrators allows rapid and complete elimination both the co-solvents as well as non-encapsulated drugs and full recovery of drug loaded ferritin nanocages. The ferritin-drug complex thus obtained was then flash freezed in liquid nitrogen and lyophilised.

[0172] Depending on the choice of co-solvent and on the intrinsic chemical properties of the drug molecule, it can be estimated that up to 150-180 drug molecules can be entrapped/adsorbed within the 24-mer ferritin cage.

[0173] Labels may also be covalently coupled to ferritin amino acid side chains (lysines or cysteines) by appropriate choice of phenylhydrazone, succinimide or maleimide activated labesl. Accordingly, i) phenylhydrazone derivative may breaks and liberates the label from the ferritin surface, ii) lysine bound derivatives may become active after full protein degradation into aminoacids or iii) cysteine bound derivative may be liberated within the cell through reductive hydrolysis of the maleimede thioether link.

Example 6—Preparation of Haemoglobin-Compound Complex

[0174] Human haemoglobin is prepared from fresh red cells as described in Rossi-Fanelli et al. (Archives of biochemistry and biophysics 77:478-492, 1958). Shortly, the heparinized blood, obtained from healthy donors, was centrifuged at 1600 rpm for 30 minutes (4° C.) to sediment the RBCs. Buffy coat was accurately removed by needle aspiration on the surface of the pellet. The plasma supernatant was discarded and the RBC pellet was washed three times by resuspending the RBCs in isotonic 0.9% saline solution and centrifuging at 1600 rpm for 30 minutes at 4° C. After the final saline wash and centrifugation step, the RBC pellet was resuspended in distilled water buffered at pH 7.2 with 5 mM potassium phosphate buffer (PB, pH=7.2) and allowed to lyse at 4° C. overnight under gentle stirring. Dialyzed RBC lysate was subsequently centrifuged at 13.000 rpm for 30 min at 4° C. and supernatant was directly loaded on an ÄKTA Explorer system equipped with an XK 26/40 column packed with Q-sepharose XL resin (GE Healthcare) at room temperature. Columns were equilibrated with buffer A (20 mM Tris-HCl, pH=8.2) at a flow rate of 12 mL/min and washed three times with the same buffer. A linear gradient elution was generated by changing from 100% buffer A to 75% buffer B (20mM Tris-Cl, plus 0.2 M NaCl pH8.20) followed by a step gradient of 100% buffer B. Upon elution, a fraction collector was used to collect protein fractions. Protein thus obtained was analyzed by SDS page and stored frozen at −80 ° C.

[0175] Human Haemoglobin (SEQ ID NO: 18 or 22, see FIG. 1) can be readily covalently linked to appropriate drug conjugates, host hydrophobic drug molecules within the heme binding pocket or even transport small cytotoxic molecules linked to the heme iron. Hb can be easily modified by selective attachment of the appropriate drug conjugate to the cysteine residue in position 93 of the beta chains, the only titratable cystein on the protein surface. The apo-protein solution must be kept at ice bath for the duration of the reconstitution process. A 1.5-fold molar excess of CuT-CPP (Cu-TCPP 4,4′,4″,4′″-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)—Cu.sup.67) in 0.1 M NaOH must be added dropwise to the apo-globin solution in 0.2 M KPi buffer at pH 7.0 vortexed quickly at room temperature and then placed back in ice bath for 30 minutes. The protein solution must then be filtered in a syringe filter before use.

Example 7—Preparation of Transferrin-Compound Complex

[0176] The serum was obtained from healthy donor and excess iron was added in the presence of citrate ions as a chelator and bicarbonate, which is facilitates for iron binding to transferrin. The reaction mixture contained 6.5 mg sodium bicarbonate and 153.16 ferric citrate in pH=8, 4° C., 1 h per 100 mL of serum. Albumin was subsequently precipitated by Rivanol (4%) by adding the alcohol solution to the serum sample in a 3.5 V/V ratio at 4° C., and pH=9.4 for 2 h. Then, the solution was centrifuged at 3000 rpm for 20 min and finally filtered by filter on a 0.8 mm syringe filter. Excess Rivanol was subsequently removed by gel-filtration on a Sephadex G-25 column in ammonium sulfate 0.025 M. A first precipitation of by saturated ammonium sulfate 50% at pH=6.5 was subsequently carried out followed by centrifugation at 3000 rpm for 10 min (immunoglobulin removal). A second precipitation at 80% saturated ammonium sulfate was then carried out thus allowing recovery of transferrin the precipitate. Solid precipitate was then dissolved in buffer of 0.06 M Tris HCl buffer, pH=8, containing 1 M NaCl. The solution was dialyzed in the same buffer to allow full removal of ammonium sulfate. Protein solution was then concentrated with a centricon PM50 centrifugal concentrator up to 10-15 mg/ml (as estimated by Bradford method) and loaded on a Sephadex G-100 gel-filtration column (2.4×80 cm) equilibrated in 1M NaCl, flow rate of 15 ml/h. Transferrin thus obtained was estimated to be 88-90% pure by SDS page. Ion-exchange chromatography by anion exchanger DEAE Sephadex A-50 was then used as a final polishing step. The transferrin sample was loaded in the column equilibrated with 0.06 M Tris HCl at pH=8 and eluted by a linear concentration gradient with elution buffer, 0.3 M Tris HCl, pH=8. Protein purity was higher than 98% with a yield of about 150 mg per 100 mL of serum.

[0177] Human Holo-transferrin, (SEQ ID NO: 28, FIG. 1) similarly to haemoglobin can be readily covalently linked to appropriate label conjugates. The apo-protein solution must be kept at ice bath for the duration of the reconstitution process. A 1.5-fold molar excess of CuT-CPP (Cu-TCPP 4,4′,4″,4′″-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)—Cu.sup.67) in 0.1 M NaOH must be added dropwise to the apo-globin solution in 0.2 M KPi buffer at pH 7.0 vortexed quickly at room temperature and then placed back in ice bath for 30 minutes. The protein solution must then be filtered in a syringe filter before use.

Example 8—Obtaining Ferritin Loaded Cells

[0178] Obtained cells are incubated in ferritin solution for a time and at the concentration sufficient to ensure proper ratio of ferritin/cell for their full load and also to ensure proper contrast content to obtain proper imaging). The time and concentration may vary depending on the number of molecules encapsulated/adsorbed into the ferritin cage, status of cell activation, condition and number of their intended administration.

[0179] For example, to ensure proper load with ferritins, cells are incubated for 1-4 hrs in ferritin solution 0.2 mg/ml in standard culture conditions. The frame of ferritin concentration may vary at least between 0.01 and 4 mg/ml as well as incubation time (5 min-6 hrs or more). Adjusting time and concentration of ferritin load to cells, the influence of ferritin and treatment conditions on cell viability should be minded. Cells obtained as stated above very easily uptake ferritins in a relatively short time (in minutes; FIG. 5). Once they absorb ferritins, they do not release it to the culture medium (FIG. 6).

[0180] Nevertheless, the person skilled in the art is able to re-adjust the above conditions and optimize the protocol for the own purposes in the own laboratory.

Example 9—Obtaining Haemoglobin Loaded Cells

[0181] Obtained cells are incubated in haemoglobin solution for a time and at the concentration sufficient to ensure proper ratio of haemoglobin/cell for their full load and also to ensure proper contrast content to obtain proper imaging. The time and concentration may vary depending on the number of molecules linked with the haemoglobin molecule, status of cell activation, condition and number of their intended administration.

[0182] For example, to ensure proper load with haemoglobins, cells are incubated for 1-4 hrs in haemoglobin solution 0.1 mg/ml in standard culture conditions. The frame of haemoglobin concentration may vary at least between 0.01 and 0.2 mg/ml as well as incubation time (5 min-4 hrs or more). Adjusting time and concentration of haemoglobin load to cells, the influence of ferritin and treatment conditions on cell viability should be minded. Cells obtained as stated above very easily uptake haemoglobins in a relatively short time (in minutes; FIG. 5).

[0183] Nevertheless, the person skilled in the art is able to re-adjust the above conditions and optimize the protocol for the own purposes in the own laboratory.

Example 10—Obtaining Transferrin Loaded Cells

[0184] Obtained cells are incubated in transferrin solution for a time and at the concentration sufficient to ensure proper ratio of transferrin/cell for their full load and also to ensure proper contrast content to obtain proper imaging. The time and concentration may vary depending on the number of molecules linked with the transferrin molecule, status of cell activation, condition and number of their intended administration.

[0185] For example, to ensure proper load with transferrins, cells are incubated for 1-4 hrs in transferrin solution 0.1 mg/ml in standard culture conditions. The frame of transferrin concentration may vary at least between 0.01 and 0.2 mg/ml as well as incubation time (5 min-4 hrs or more). Adjusting time and concentration of transferrin load to cells, the influence of transferrin and treatment conditions on cell viability should be minded. Cells obtained as stated above very easily uptake transferrin in a relatively short time (in minutes; FIG. 5).

[0186] Nevertheless, the person skilled in the art is able to re-adjust the above conditions and optimize the protocol for the own purposes in the own laboratory.

Example 11—Cell Labeling with Radioisotope

[0187] In this invention cells were labeled with .sup.18F-FDG in order to become imaged on PET. Cells have been detached from the plate and incubated with .sup.111F-FDG solution in adequate concentration to ensure the most optimal .sup.18F-FDG uptake by cells allowing their radio-detection at the site of their accumulation. In this practical invention cells were pre-treated with various cytokine cocktails, growth factors and other factors influencing their activation state to enhance their uptake of label and incubated in the warm .sup.18F-FDG solution containing 20-60 MBq. After labeling, cells should be centrifuged and supernatant should be removed. This step should be repeated until no radioactivity is detected in the supernatant.

Example 12—Ferritin/Haemoglobin/Transferrin/Label-Leukocyte Complex as Useful Delivery Tool to Tumor

[0188] The macrophages from Example 1 prepared as described in Examples 8, 9, 10 and 11; monocytes from Example 2 prepared as described in Examples 8, 9, 10 and 11, granulocytes from Example 3 prepared as described in Examples 8, 9, 10 and 11, and lymphocytes from Example 4 prepared as described in Examples 8, 9, 10 and 11, very easily transport ferritins, haemoglobins, transferrins and labels to the tumor in the enough amount to be detected using imaging systems (FIGS. 2, 3, 4, 7, and 8).

Example 13—Leukocyte-Protein Carrier Complex as Useful Targeted Drug Delivery Agent to Hypoxic Regions

[0189] Macrophages prepared as above are injected into the tail vein of animal with the tumour (appropriate number of macrophages should be adjusted to the tumour size, stage of development and presence of metastases). As it is shown on FIGS. 2, 3, 4, 7, 8 and 10 they specifically reach the tumour (after a few hrs) and also disperse in other organs of the whole animal (. Moreover, as it is shown on FIG. 9, in hypoxic model they are also able to migrate to the avascular and hypoxic sites.

[0190] For the imaging purposes, 1-50 millions of macrophages were injected into the tail vein of mammary or colon cancer tumour-bearing animal. Before, macrophages were pre-labeled with Cell Tracker and loaded with ferritin-FITC (as shown in Example 8). Using two-photon imaging of the tumour mass 8 hrs after administration of macrophages the presence of macrophages carrying Ferritin-FITC was detected (FIG. 7). Their specific targeting of tumour but also their migration to other organs was shown using whole animal body imaging (IVIS) after macrophage pre-labeling using XenoLight DiR lipophilic DIR cytoplasmic dye (FIG. 8).

Example 14—Leukocyte-Protein Carrier Complex or Labeled Leukocyte as Useful Imaging Tool

[0191] The targeted delivery system described in present invention constitutes very useful imaging tool. As it is presented on FIGS. 4 and 10, after injection of 1-50 ml of macrophages loaded with ferritin coupled as described in Example 8 with a contrast agent (in this example: ferrihydrite, however the same results are obtained with isotope, e.g. .sup.123I) or labeled with isotope (in this case .sup.18F-FDG) (FIG. 10) they can be easily detected by MRI, PET or SPECT. In this example (FIG. 4), mammary-tumour bearing mice were imaged using MRI at 3, 22 and 24 hours after i.v. injection of macrophages loaded with ferritin Fh. As it is shown on FIG. 4 the ferritin/macrophage complex is very useful imaging tool. The mouse was treated (at time point 0 h) with macrophages. Then increased diameter of blood vessels (arrow) filled with injected macrophages (giving significant T2-signal reduction) has been observed and afterword macrophages spread to the tissue (spot-like pattern; arrows). These changes (in the same time points) were observed in all examined mice.

[0192] Macrophages were also labeled with .sup.18F-FDG (1-50 min) and imaged using PET at 1 h after i.v. administration to the tumour-bearing mice. These mice were inoculated with 4T1 metastatic cell line 3 weeks before the experiment and metastases in the lungs, liver and spleen were histopatologically confirmed. At FIG. 10 it is seen that macrophages migrated to the regions with metastatic tumours allowing their visualization at PET.

[0193] While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention.

[0194] The present invention also relates to the following aspects and preferred embodiments of these aspects. The definitions provided above similarly apply to below aspects and embodiments. [0195] 1. Targeted delivery system comprising an activated macrophage loaded with ferritin carrying a contrast agent. [0196] 2. The targeted delivery system according to embodiment 1, wherein the contrast agent is a ferrihydride or an isotope. [0197] 3. Method of preparation of the targeted delivery system comprising an activated macrophage loaded with ferritin carrying a contrast agent comprising steps of [0198] a) ferritin purification; [0199] b) obtaining ferritin carrying a contrast agent by linking of ferritin with said contrast agent; [0200] c) activation of isolated macrophages; [0201] d) incubation of macrophages in solution of ferritin carrying a contrast agent as obtained in step b) for a time and at the ferritin concentration sufficient to ensure full load of ferritin carrying a contrast agent into macrophages. [0202] 4. The method of embodiment 3, wherein activated macrophages are bone marrow originated macrophages. [0203] 5. The method of embodiment 3, wherein activated macrophages are blood originated macrophages. [0204] 6. The method of embodiment 3, wherein activated macrophages are derived from macrophage cell lines. [0205] 7. The method of any one of embodiments 3-6, wherein activated macrophages are macrophages polarized towards M1 or M2. [0206] 8. The method of embodiment 7 wherein activated macrophages have been polarized towards M2. [0207] 9. The method of embodiment 7 wherein activated macrophages have been manipulated with respect to iron metabolism. [0208] 10. The method of any one of embodiments 3-9, wherein the contrast agent is a ferrihydride or an isotope. [0209] 11. Targeted delivery system as defined in any of embodiments 1 or 2 for use as imaging tool.

[0210] In a preferred embodiment the present invention does not comprise the subject-matter of items 1 to 11 above.