Cellular targeted pharmaceutically active substance or 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 an active pharmaceutically active substance and/or label as well as methods for producing such isolated cellular targeted delivery system and uses of such system for prophylaxis, therapy, diagnosis or theragnosis, in particular for prophylactic or therapeutic vaccination, therapy of cancer, particularly metastatic cancer or inflammatory diseases.

1 . An isolated targeted delivery system comprising a CD45 +  monocyte, CD45 +  monocyte-macrophage, CD45 +  lymphocyte and/or CD45 +  granulocyte comprising within said cell a complex of one or more iron binding proteins and a pharmaceutically active substance, label or pharmaceutically active substance and label. 
     
     
         2 . An isolated targeted delivery system comprising a CD45 +  leukocyte cell comprising one or more label, in particular radiolabels or their conjugates and combinations. 
     
     
         3 . The isolated targeted delivery system according to  claim 1 , wherein the CD45 +  leukocyte cell is producible from a CD34 +  hematopoietic precursor cell. 
     
     
         4 . The isolated targeted delivery system according to  claim 3 , wherein
 (i) the monocyte is a CD11b +  monocyte, but is not a dendritic cell which differentiation is controlled by following transcription factors: IFN-regulatory factor 8 (IRF8), nuclear factor interleukin (IL)-3-regulated protein (NFIL3), basic leucine zipper transcriptional factor ATF-like 3 (BATF3) or transcription factor RelB (RELB), Spi-1 Proto-Oncogene (PU/1), recombining binding protein suppressor of hairless (RBPJ), IFN-regulatory factor 4 (IRF4) or transcription factor E2-2 (also known as TCF4);   (ii) the differentiated monocyte is selected from the group consisting of a macrophage, an activated macrophage,   a monocyte-macrophage or an activated monocyte-macrophage expressing at least one chemokine receptor, or at least one growth factor receptor;   (iii) the lymphocyte is selected from the group consisting of a CD3 +  and CD4 +  or CD8 +  T lymphocyte, or a CD19 + , CD20 + , CD21 + , CD19 +  CD20 + , CD19 +  CD21 + , CD20 +  CD21 + , or CD19 +  CD20 +  CD21 +  B lymphocyte, and a natural killer (NK) cell; or   (iv) the granulocyte is selected from the group consisting of a neutrophil, an eosinophil and a basophil.   
     
     
         5 . The isolated targeted delivery system of  claim 4 , 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;   or with a factor capable of altering the macrophages ability to secrete cytokines;   (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.   
     
     
         6 . The targeted delivery system according to  claim 5 , wherein the factor capable of altering expression markers on macrophages is an M1 inducer or an M2 inducer, wherein:
 (i) the M1 inducer is selected from the group consisting of LPS, INF-γ, 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.   
     
     
         7 . The isolated targeted delivery system of  claim 4 , wherein the monocyte-macrophage cell:
 (i) is producible from a CD34 +  hematopoietic precursor cell;   (ii) is producible by in vitro incubation of monocytes/monocyte-macrophage with at least one inducer;   (iii) is characterized by expression of at least one of the following antigens: TfR + , CD163 + , TIM-2 + , CD14 + , CD16 + , CD33 + , and/or CD115 + ;   (iv) is characterized by expression of at least one of the following antigens: TfR + , CD163 + , TIM-2 + , CXCR4 + , CD14 + , and/or CD16 + ; and/or   (v) has the ability to phagocytose.   
     
     
         8 . The targeted delivery system according to  claim 7 , wherein the inducer is an M1 inducer or an M2 inducer, wherein:
 (i) the M1 inducer is selected from the group consisting of LPS, INF-γ, 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.   
     
     
         9 . The isolated targeted delivery system of  claim 4 , wherein the lymphocyte:
 (i) is obtainable from blood, spleen, or bone marrow or is producible from a CD34 +  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 +  and CD4 +  or CD8 +  or (b): CD19 + , CD20 + , CD21 + , CD19 +  CD20 + , CD19 +  CD21 + , CD20 +  CD21 + , or CD19 +  CD20 +  CD21 +  antigen.   
     
     
         10 . The isolated targeted delivery system of  claim 4 , wherein the granulocyte:
 (i) is obtainable from blood, spleen or bone marrow or producible from a CD34 +  precursor cell;   (ii) is characterized by expression of at least one of the following CD66b +  and/or CD193 + ;   (iii) is a polymorphonuclear leukocyte characterized by the presence of granules in their cytoplasm; and/or   (iv) is characterized by expression of at least one of the following: TfR + , CD163 + , TIM-2 + , and/or CXCR4 + .   
     
     
         11 . The isolated targeted delivery system of  claim 4 , wherein the NK cell:
 (i) is obtainable from blood, spleen or bone marrow or producible from a CD34 +  precursor cell; and/or   (ii) is characterized by the lack of CD3 expression and expression of at least one of the following CD56 +  and/or CD94 + , CD158a +  CD158f +  CD314 +  CD335 + .   
     
     
         12 . The isolated targeted delivery system of  claim 1 , wherein the iron binding protein is selected from the group consisting of ferritin haemoglobin, haemoglobin-haptoglobin complex, hemopexin, transferrin; and lactoferrin. 
     
     
         13 . The isolated targeted delivery system according to  claim 1 , wherein the pharmaceutically active substance is selected from the group consisting of a protein, a nucleic acid, a non-protein non-nucleic acid compound with a molecular weight of less than 1.5 kD; an anticancer drug; an anti arteriosclerotic drug; and anti-inflammatory drug; and photosensitizing compound; a virus; and a α or β radiation emitting radioisotope, which also emit a cell damaging amount of γ radiation, or a complex of the compound or isotope linked to a nanoparticle. 
     
     
         14 . The isolated targeted delivery system according to  claim 13 , wherein the anticancer drug is selected from the group consisting of an apoptosis-inducing drug, an alkylating substance, anti-metabolites, antibiotics, epothilones, nuclear receptor agonists and antagonists, an anti-androgene, an anti-estrogen, a platinum compound, a hormone, an antihormone, an interferon, an inhibitor of cell cycle-dependent protein kinases (CDKs), an inhibitor of cyclooxygenases and/or lipoxygenases, a biogeneic fatty acid, a biogenic fatty acid derivative, prostanoids, leukotrienes, an inhibitor of protein kinases, an inhibitor of protein phosphatases, an inhibitor of lipid kinases, a platinum coordination complex, an ethyleneimine, a methylmelamine, a triazine, a vinca alkaloid, a pyrimidine analog, a purine analog, an alkylsulfonate, a folic acid analog, an anthracendione, a substituted urea, a methylhydrazin derivative, an ene-diyne antibiotic, a maytansinoid an auristatine derivate, immune check-point inhibitor, and an inhibitor of tumour-specific protein or marker. 
     
     
         15 . The isolated targeted delivery system according to  claim 13 , wherein the anticancer drug is a acediasulfone, aclarubicine, ambazone, aminoglutethimide, L-asparaginase, azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chlorambucil, cis-platin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin dapsone, daunorubicin, dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate, estrogen, ethinylestradiole, etoposide, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide fosfestrol, furazolidone, gemcitabine, gonadotropin releasing hormone analog, hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin, hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon α, irinotecan, leuprolide, lomustine, lurtotecan, mafenide sulfate olamide, mechlorethamine, medroxyprogesterone acetate, megastrolacetate, melphalan, mepacrine, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, nifuratel, nifuroxazide, nifuralazine, nifurtimox, nimustine, ninorazole, nitrofurantoin, nitrogen mustards, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman, prednimustine, prednisone, preussin, procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustine streptozocine, sulfacarbamide, sulfacetamide, sulfachlopyridazine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine, sulfaguanole, sulfamethizole, sulfamethoxazole, co-trimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin, sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen, taxol, teniposide, tertiposide, testolactone, testosteronpropionate, thioguanine, thiotepa, tinidazole, topotecan, triaziquone, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinblastine, vinorelbine, and zorubicin. 
     
     
         16 . The isolated targeted delivery system according to  claim 13  wherein the immunomodulatory drugs activate or inhibit activity of immune cells. 
     
     
         17 . The isolated targeted delivery system according to  claim 13 , wherein the anticancer drug is a proliferation inhibiting protein, or an antibody or antibody like binding protein that specifically binds to a proliferation promoting protein or a nucleic acid. 
     
     
         18 . The isolated targeted delivery system according to  claim 1 , wherein the pharmaceutically active substance is a hypoxia-activated prodrug. 
     
     
         19 . The isolated targeted delivery system according to  claim 1 , wherein the pharmaceutically active substance is an antigen or a nucleic acid encoding an antigen. 
     
     
         20 . The isolated targeted delivery system of  claim 1 , 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. 
     
     
         21 . The isolated targeted delivery system of  claim 1 , wherein the label comprises a chelating agent which forms a complex with divalent or trivalent metal cations. 
     
     
         22 . The isolated targeted delivery system of  claim 21 , 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). 
     
     
         23 . The isolated targeted delivery system of  claim 14 , wherein the contrast agent comprises a paramagnetic agent. 
     
     
         24 . The isolated targeted delivery system of  claim 20 , wherein the radioisotope/fluorescence emitting isotope is selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as  65 Tb, fluorescence emitting isotopes, such as  18 F,  51 Cr,  67 Ga,  68 Ga,  89 Zr,  111 In,  99mTc ,  140 La,  175 Yb,  153 Sm,  166 Ho,  88 Y,  90 Y,  149 Pm,  177 Lu,  47 Sc,  142 Pr,  159 Gd,  212 Bi,  72 As,  72 Se,  97 Ru,  109 Pd,  105 Rh,  101m15 Rh,  119 Sb,  128 Ba,  123 I,  124 I,  131 I,  197 Hg,  211 At,  169 Eu,  203 Pb,  212 Pb,  64 Cu,  67 Cu,  188 Re,  186 Re,  198 Au and  199 Ag as well as conjugates and combinations of above with proteins, peptides, small molecular inhibitors, antibodies or other compounds. 
     
     
         25 . The isolated targeted delivery system of  claim 20 , wherein the fluorescence dye is 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. 
     
     
         26 . The isolated targeted delivery system according to  claim 20 , wherein the detectable polypeptide is an autofluorescent protein. 
     
     
         27 . The isolated targeted delivery system according to  claim 1 , wherein:
 (i) the bond(s) between the iron binding protein(s) and the pharmaceutically active substance, label or pharmaceutically active substance and label comprised in the complex are covalent and/or non-covalent; and/or   (ii) the pharmaceutically active substance, label or pharmaceutically active substance and label comprised in the complex is entrapped/encapsulated by the iron binding protein or multimers thereof.   
     
     
         28 . Method of preparation of the isolated targeted delivery system of  claim 1  comprising steps of
 a) providing purified iron binding protein; 
 b) covalently or non-covalently linking a pharmaceutically active substance, label or pharmaceutically active substance and label to and/or encapsulating a pharmaceutically active substance, label or pharmaceutically active substance and label in an iron binding protein; 
 c) providing a CD45 +  leukocyte cell; and 
 d1) incubating the CD45 +  leukocyte cell in the presence of the iron binding protein produced in step b) until the CD45 +  leukocyte cell is at least partially loaded with the complex of the iron binding protein and the a pharmaceutically active substance, label or pharmaceutically active substance and label produced in step b); and/or 
 d2) incubating CD45 +  leukocyte cell in the presence of the label until the CD45 +  leukocyte cell is at least partially labelled with the label. 
 
     
     
         29 . The isolated targeted delivery system of  claim 1  or producible according to the method of  claim 28  for use as a medicament or diagnostic. 
     
     
         30 . A pharmaceutical composition comprising the isolated targeted delivery system of  claim 1  and a pharmaceutically acceptable carrier and/or suitable excipient(s). 
     
     
         31 . A method for preventing, treating or diagnosing a tumour, an inflammatory disease or ischemic areas, or for prophylactic of therapeutic vaccination, comprising administration of the isolated targeted delivery system of  claim 1  in an effective amount to a patient in need thereof.