EP1474519A2 - Mikroorganismus zur gentherapeutischen behandlung proliferativer erkrankungen - Google Patents

Mikroorganismus zur gentherapeutischen behandlung proliferativer erkrankungen

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Publication number
EP1474519A2
EP1474519A2 EP03704314A EP03704314A EP1474519A2 EP 1474519 A2 EP1474519 A2 EP 1474519A2 EP 03704314 A EP03704314 A EP 03704314A EP 03704314 A EP03704314 A EP 03704314A EP 1474519 A2 EP1474519 A2 EP 1474519A2
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EP
European Patent Office
Prior art keywords
microorganism
tumor
cell
expression
component
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP03704314A
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German (de)
English (en)
French (fr)
Inventor
Werner Goebel
R. Ulf Rapp
Hans-Harald Sedlacek
Joachim Fensterle
Ivaylo Gentschev
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aeterna Zentaris GmbH
Original Assignee
Zentaris AG
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Application filed by Zentaris AG filed Critical Zentaris AG
Publication of EP1474519A2 publication Critical patent/EP1474519A2/de
Withdrawn legal-status Critical Current

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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01031—Beta-glucuronidase (3.2.1.31)
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/16—Exonucleases active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters (3.16)
    • C12Y301/16001—Spleen exonuclease (3.1.16.1), i.e. 5->3 exoribonuclease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the invention relates to a microorganism with foreign nucleotide sequences, by means of which expression products having an antiproliferative or cytotoxic effect can be expressed, and the use of such microorganisms for the production of pharmaceutical compositions, a plasmid and a method for producing such a microorganism, and uses of such microorganisms.
  • Microorganisms reduced in their virulence such as genetically modified viruses or bacteria attenuated in their virulence, are becoming increasingly important as carriers of foreign nucleic acid sequences in the context of gene therapy.
  • the foreign nucleic acids are either introduced into tissue cells in vitro and these cells are administered to the patient, or the microorganisms are injected into the patient in the expectation that the microorganisms, as gene transporters, will transfer the foreign nucleic acid into the desired tissue cell.
  • Microorganisms are particles. After injection into an organism, these particles are mainly absorbed by the so-called reticuloendothelial system. In order to achieve an enrichment of the microorganisms used as gene transporters in a target tissue despite this elimination mechanism, the microorganisms were equipped with cell-specific ligands. So far, the elimination of the Microorganisms are only slightly reduced by the reticuloenothelial system. -
  • a key research goal of gene therapy is the therapy of proliferative diseases, such as tumors, leukemias, chronic inflammation, autoimmune diseases and rejection of transplanted organs, the treatment of which, despite all the successes in drug therapy, is still inadequate.
  • proliferative diseases such as tumors, leukemias, chronic inflammation, autoimmune diseases and rejection of transplanted organs
  • the treatment of which, despite all the successes in drug therapy, is still inadequate for example, despite all the successes of surgery, radiotherapy, chemotherapy and also immunotherapy in the treatment of tumors, it has so far not been possible to cure advanced tumors of the head and neck, the central nervous system, the mammary gland, the lungs, and the gastrointestinal tract , the liver, the pancreas, the kidney, the skin, the ovaries and the prostate.
  • the reasons for this inadequate success of tumor therapy are varied and not yet fully known.
  • the main reasons include i) pre-existing (primary) resistance of the tumor cells to the concentrations of chemotherapeutic agents, radiation or immunotherapeutic agents that can be achieved in vivo, ii) resistance to the respective therapeutic agent that arises in response to the therapy.
  • tumor therapeutics include, iv) an excessively high volume of distribution, v) inadequate accumulation on the tumor or on the tumor cells, vi) inadequate penetration ability in the tumor and / or vii) the toxic effect on the entire organism, which is a Increased dose limits for increased tumor accumulation.
  • Tumor cell-specific ligands for example antibodies or their cleavage products, coupled to cytostatics, to antitumoral cytokines, to cytotoxic proteins, or to isotopes did indeed lead to an accumulation of the cytotoxic active substances on the tumor compared to
  • amplification systems were designed with the help of which the concentration of the respective active substance on the tumor could be increased.
  • the aim of an amplification system was to introduce into the tumor those enzymes which were not generally accessible or foreign in the rest of the body and which in turn could convert or split a non-toxic precursor of a cytostatic into the cytotoxically active cytostatic in the tumor.
  • the enzymes were introduced into the tumor either by the administration of tumor cell-specific ligands coupled to these enzymes (for example in the form of Antibody-Derived Enzymemediated Prodrug Therapy; ADEPT) or by Administration of genes for these enzymes with the aid of tumor cell-specific or non-specific vectors (Gene Derived Enzyme-Mediated Prodrug Therapy; GDEPT) (Sed- lacek et al., Contributions to Oncology 43: 1-145, 1992; Sedlacek, Critical Reviews in Oncology / He atology 37: 169-215, 2001; McCormick Nature Reviews Cancer 1: 130-141,2001; Carter, Nature Reviews Cancer 1: 18-129,2001).
  • GDEPT Gene Derived Enzyme-Mediated Prodrug Therapy
  • Another amplification system is based on the induction of an immune reaction against tumor cells, in the course of which specific antibody-forming cells and cytotoxic cells proliferate.
  • Tu or ⁇ ntigen are administered in a suitable preparation. The aim is to break the immune tolerance that is evident in tumor patients to his tumor and / or resistance of his tumor to his own immune response.
  • a technique has been developed to express expression products of nucleic acid sequences introduced into bacteria on the cell membrane of these bacteria or to have them secreted.
  • This technique is based on the Escherichia coli hamolysin system HlyAs, which is the prototype of a type I secretion system for Gram-negative bacteria.
  • HlyAs secretion vectors were developed that enable protein antigens to be efficiently discharged into Samonella enterica, Yersinia enterocolitica and Vibrio cholerae.
  • Such secretion vectors contain the cDNA of any protein antigen coupled to the nucleotide sequence for the HlyA signal peptide, for the Hamolysm secretion apparatus, hlyB and hlyD and the hly-specific promoter. With the help of this secretion vector, a protein can be expressed on the surface of this bacterium.
  • Such genetically modified bacteria induce as vaccines a much stronger immune protection than bacteria in which the protein expressed by the introduced nuclear acid remains inside the cell (Donner et al EP 1015023 A, Gentschev et al, Gene, 179: 133-140, 1996; Vac - cine 19: 2621-2618, 2001, Hess et al PNAS 93: 1458-1463, 1996).
  • the disadvantage of this system is that the amount of the protein expressed on the outer surface of the bacterium is extremely small due to the use of the hly-specific promoter.
  • Mammalian cells from carrier bacteria such as Salmonella and Listeria monocytogenes have been developed. Genes contained in these plasmids could also be expressed in the mammalian cells if they were under the control of a eukaryotic promoter. Plasmids were introduced into Listeria monocytogenes germs which contain a nucleotide sequence for any antigen under the control of any eukaryotic promoter.
  • Virulence-attenuated variants of bacteria that colonize intracellularly have been developed.
  • Listeria monocytogenes, Salmonella enterica sv. Typhimuriu and Typhi, as well as BCG such variants are already used as well-tolerated live vaccines against typhoid and tuberculosis.
  • These bacteria, including their attenuated mutants, are generally immunostimulatory and can trigger a good cellular immune response.
  • L stimulates. monocytogenes particularly through the activation of TH1 cells the proliferation of cytotoxic lymphocytes.
  • These bacteria deliver secreted antigens directly into the cytosol Antigen-presenting cells (APC; macrophages and dendritic cells), which in turn express the costing molecules and trigger an efficient stimulation of T cells.
  • APC cytosol Antigen-presenting cells
  • the Listeria are partially broken down in phagosomal compartments and the antigens produced by these carrier bacteria can therefore be presented on the one hand via MHC class II molecules and thus lead to the induction of T helper cells.
  • the listeria replicate in the cytosol of APCs; Antigens produced and secreted by these bacteria are therefore preferably presented via the MHC class I route, which induces CTL responses against these antigens.
  • Virulence-attenuated Salmonella enterica strains into which nucleotide sequences coding for tumor antigens had been introduced, were able to provide specific protection against different experimental tumors as oral antigen-expressing bacterial carriers (Medina et al., Eur J Immunol 30: 768- 777, 2000, Zoller and Christ J Immunol 166: 3440- 3450, 2001; Xiang et al. , PNAS 97: 5492-5497, 2000).
  • Recombinant SaJmonelJa strains were also effective as a prophylactic vaccine against viral infections (HPV) infections (Benyacoub et al., Infect Immun 67: 3674-3679, 1999) and for the therapeutic treatment of a mouse tumor immortalized by a tumor virus (HPV) (Revaz et al., Virology 279: 354-360, 2001).
  • HPV viral infections
  • Salmonella strains were selected that colonize specifically selected tumor tissues (Murray et al j Bacteriol 183: 5554-5564, 2001).
  • the invention is based on the technical problem of specifying a pharmaceutical composition which has an increased effectiveness in the treatment of proliferative diseases, in particular in tumor therapy.
  • the invention teaches a coated microorganism, in the genome of which the following components are inserted and expressible: I) a nucleotide sequence coding for a direct or indirect, antiproliferative or cytotoxic expression product or for several different ones
  • Expression products II) a nucleotide sequence which codes for a blood plasma protein under the control of an activation sequence which can be activated in the microorganism or which is constitutively active, III) optionally, a nucleotide sequence which codes or constitutes for a cell-specific ligand under the control of an activation sequence which can be activated in the microorganism is active, IV) a nucleotide sequence for a transport system which the expression of the expression products of components I) and II) and optionally III) on the outer surface of the microorganism or the secretion of the expression products of component I) and expression of component II) and optionally III) and preferably constitutively active, V) optionally a nucleotide sequence for a protein for lysing the microorganism in the cytosol of mammalian cells and for the intracellular release of plasmids with at least one or more of the components I) and VI) contained in the lysed micro organism, and VI) an activation sequence which can be
  • coated microorganisms are preferably described as carriers for genetic information and the use of these covered microorganisms for the prophylaxis and therapy of a proliferative disease.
  • the invention is based on the following experiences and technical developments.
  • the invention thus preferably relates to coated microorganisms as carriers for nucleotide sequences for the treatment of proliferative diseases, the following components having been inserted into the microorganisms: I) at least one nucleotide sequence, coding for at least one directly or indirectly antiproliferative or cytotoxic expression product, II) at least one nucleotide sequence which codes for at least one blood plasma protein under the control of at least one activation sequence which can be activated in the microorganism, III) optionally at least one nucleotide sequence which codes for at least one cell-specific ligand under the control of at least one activation sequence which can be activated in the microorganism, IV) at least one nucleotide sequence for at least one transport system, which the expression of the expression products of components I), II) and III) on the outer surface of the microorganism or the secretion of the compo components I), allows II) and III), V) optionally at least one nucleotide sequence for minde ⁇ least one protein
  • Component I is at least one nucleotide sequence, coding for at least one expression product which has a direct or indirect antiproliferative or cytotoxic expression product.
  • Expression products having a direct antiproliferative effect in the sense of the invention are, for example, interferons such as, for example, IFN-alpha, IFN-gamma, IFN- ⁇ , interleukms which inhibit immune cells or tumor cells, such as IL-10, IL-12, proapoptotic peptides or proteins such as for example TNF-alpha, FAS ligand, TNF-related apoptosis inducing ligand (TRAIL), antibodies or fragments of antibodies which have an inhibitory or cytotoxic effect on an immune cell, a tumor cell, or a cell of the tissue from which the tumor originates How, for example, antibodies directed against l) a tumor-associated or tumor-specific antigen, n) an antigen on lymphocytes, such as, for example, against the T cell receptor, the B cell
  • the receptor for an interleukm such as IL -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15 or -16
  • the receptors for an interferon or the receptor f r a chemokm for example for RANTES, MCAF, MlP-alpha, MlP-ß, IL-8, MGSA / Gro, NPA-2 or IP-10
  • a tissue-specific antigen such as, for example, against a tissue-specific antigen of the cells of the mammary glands , Kidneys, birthmarks, prostate, thyroid gland, gastric mucosa, ova, cervix, bladder mucosa
  • Proteins with an indirect antiproliferative effect are, for example, inducers of acute inflammation and immune reactions, such as, for example, chokines such as RANTES (MCP-2), monocyte chemotactic and activating factor (MCAF), IL-8, macrophage inflammatory protein-1 (MIP-l-alpha, -ß), neutrophil activating protein-2 (NAP-2), interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, human leukemia inhibitory factor (LIF), IL- 6, IL-7, IL-9, IL-11, IL-13, IL-14, IL-15, IL-16, cytokines such as GM-CSF, G-CSF, M-CSF, enzymes for activating or cleaving the inactive precursor of a cytotoxic substance into a cytotoxic substance, these enzymes being an oxidoreductase, a transferase, a hydrolase or a lyase.
  • Examples of such enzymes are ⁇ -glucuronidase, ⁇ -galactosidase, glucose oxidase, glycosidase, alcohol dehydrogenase, lactoperoxidase, urokinase, tissue plasminogen activator carboxy peptidase, cytosine deainase, deoxycytidine kinase, lipase, thymidine Acid phosphatase, alkaline phosphatase, kinase, purine nucleoside phosphorylase, glucose oxidase, lactoperoxidase, lactaxoxidase, Penicillm-V-amidase, penicillin-G-aidase, lysozyme, ⁇ -lactamase, A mopeptidase, carboxypeptidase A, B or G2, nitroreductase, cytochrome p450 oxidase.
  • the enzyme can be derived from a virus, a bacterium, a yeast, a mollusk, an insect or a nipple. Enzymes which are derived from humans are preferably used.
  • nuclear acid constructs which code for a fusion product of a cell-specific ligand with an enzyme and / or proteins which inhibit angiogenesis, for example plasmmogenactivatonn-h ⁇ b ⁇ tor-1 (PAI-1); PAI-2 or PAI-3, angiostat or endostat, interferon -alpha, -ß, or -gamma, interleukm 12, platelet factor 4, thrombospondm -1 or -2, TGF-ß, TNF-alpha, vascular endothelial cell growth Inhibitor (VEGI).
  • PAI-1 plasmmogenactivatonn-h ⁇ b ⁇ tor-1
  • PAI-2 or PAI-3 angiostat or endostat
  • interferon -alpha interferon -alpha
  • component I) can represent one or more nucleotide sequences coding for one or more identical or different, directly or indirectly antiproliferative or cytotoxic proteins. Combinations of proteins which have an additive or synergistic effect are preferred. Additive or synergistic effects are to be expected, for example, in the following combinations of unequal proteins: cytotoxic proteins and proapoptotic proteins, enzymes and cytotoxic and / or proapoptotic proteins, anti-angiogenic proteins and cytotoxic and / or proapoptotic proteins, inducers of inflammation and enzymes or cytotoxic, proapoptotiscne and / or antiangiogenic proteins.
  • Component II is a nucleotide sequence which for at least one blood plasma protein is under the control of an activatable in the microorganism Activation sequence encoded.
  • Human blood plasma proteins are preferred, specifically those which have an average blood residence time of more than 24 hours. These include in particular, for example, albumin (nucleotides 1-2258, Hinchliffe et al, EP 0248637-A, December 9, 1987).
  • Component III) is a nucleotide sequence which codes for a cell-specific ligand under the control of an activation sequence which can be activated in the microorganism.
  • this ligand depends on the type of proliferative disease, for which the microorganism is used and the cells or the tissue with which component I) is to be brought into contact in the microorganism in order to achieve the therapeutic effectiveness.
  • ligands with specificity for tumor cells i.e.
  • tumor-associated or tumor-specific antigens or tumor endothelial cells or for tissue cells from which the respective tumor originates are used for tumor diseases, for example for cells of the thyroid gland, the prostate, the ovary, the mammary gland, the kidney Gastric mucosa, birthmarks, cervix, bladder; in chronic inflammation, cellular autoimmune diseases and rejection of transplanted organs, ligands with either specificity for macrophages, dentritic cells, T-lymphocytes or for activated endothelial cells.
  • Such ligands are, for example, specific antibodies, or antigen-binding fragments of these antibodies, growth factors, interleukins, cytokines or cell adhesion molecules which are present on tumor cells, on leukemia cells, on tumor endothelial cells, on tissue cells, on macrophages, dentritic cells, T-lymphocytes or on activated endothelial cells bind selectively.
  • Component IV) is a nucleotide sequence coding for a transport system which pression the explosion of the expression products of components I), II) and / or III) to the outer surface of the microorganism he ⁇ made possible.
  • the respective component can either be secreted either selectively or be expressed on the membrane of the microorganism, ie expressed at the membrane.
  • Components II) and III) are preferably expressed in the membrane.
  • Such transport systems are for example Hämolysintransportsignal of E. coli (Nukleotidsequen ⁇ zen containing HlyA, HlyB and HlyD under the control of hly-specific promoters, Gentschev et al Gene, 179: 133-140, 1996).
  • the following transport signals can be used: the C-terminal HlyA transport signal for secretion, in the presence of HlyB and HlyD proteins; for the membrane-permanent expression the C-terminal HlyA transport signal, in the presence of the HlyB protein; the hemolysin transport signal from E.
  • Component V) is a nucleotide sequence coding for at least one lytic protein which is expressed in the cytosol of a mammalian cell and which lyses the microorganism to release the plasmids in the cytosol of the host cell.
  • lytic proteins endolysins
  • endolysins are, for example, listeria-specific lysis proteins such as PLY551 (Loessner et al Mol Microbiol 16: 1231-41, 1995), the Listeria-specific holin under the control of a listerial promoter.
  • a preferred embodiment of this invention is the combination of different components nation ⁇ V), for example the combination of a lysis protein with a holin.
  • Component VI) represents any activator sequence which controls the expression of component I).
  • component VI) is one of the activation sequences which are known to the person skilled in the art and can be activated in the bacterium.
  • Activation sequences of this type are, for example, constitutively active promoter regions, such as the promoter region with the “ribosomal binding site” (RBS) of the beta-lactamase gene from E. coli or the tetA gene (Busby and Ebright, Cell 79: 743-746., 1994 ), inducible promoters, preferably promoters that become active after being taken up into the cell.
  • the latter includes the actA promoter from L.
  • Activator sequences which are activated in this cell after release of the plasmids of the bacterial carrier in the cytosol of the target cell are preferred.
  • the CMV enhancer, the CMV promoter, the SV40 promoter or any other promoter or enhancer sequence known to the person skilled in the art can be used.
  • Cell-specific or function-specific activator sequences are also preferred.
  • the choice of the cell-specific or function-specific activator sequence depends on the cell or the tissue in which the bacterial carrier or the plasmids released from the bacterial carrier are to express component I).
  • Such activator sequences are, for example, tumor cell-associated activator sequences (these include activator sequences of the genes for Midkine, GRP, TCF-4, MUC-1, TERT, MYC-MAX, surfactant protein, alpha-fetoprotein, CEA, tyrosinase, fibrillary acidic protein, EGR -1, GFAP, E2F1, basic myelin, alpha-lactalbumin, osteocalcin, thyroglobulin and PSA (McCormick Nature Reviews Cancer 1: 130-141,2001)), endothelial cell-specific activator sequences (these include activator sequences of the genes for proteins derived from Endothelial cells are preferentially expressed (Sedlacek, Critical Reviews in Onology / Hematology 37: 169-215,
  • Kidney activator sequences of the genes for proteins which are expressed in macrophages, dendritic cells or lymphocytes such as interleukins, cytokines, chemokines, adhesion molecules, interferons, receptors for interleukins, cytokines, chemokines, or interferons, activator sequences which activate in hypoxia such as the activator sequence for VEGF or for erythropoietin.
  • Components I) to VI) are inserted into the microorganisms using the molecular biological methods known to the person skilled in the art.
  • the person skilled in the art is familiar with how the components are inserted into suitable plasmids and how these plasmids are introduced into the bacteria.
  • these microorganisms are administered to a patient for the prophylaxis or therapy of a proliferative disease such as, for example, a tumor, leukemia, chronic inflammation, an autoimmune disease or rejection of an organ transplant.
  • the microorganisms according to the invention are administered locally or systemically in a suitable preparation, for example in the bloodstream, in a body cavity, in an organ, in a joint or in the connective tissue.
  • a suitable preparation for example in the bloodstream, in a body cavity, in an organ, in a joint or in the connective tissue.
  • the suspension and incubation is preferably carried out in solutions of substances or solutions of mixtures of substances which have a long blood residence time.
  • substances include, for example, albumin, transferrin, prealbumin, hemoglobin, haptoglobin, alpha-1 lipoprotein, alpha-2 lipoprotein, ⁇ -1 lipoprotein, alpha-2 macroglobulin, polyethylene glycol (PEG), conjugates of PEG with natural or synthetic polymers, such as with polyethyleneimines, dextrans, polygelines, hydroxyethyl starch.
  • the suspension and incubation in such a solution cause the substances to be adsorbed onto the surface of the microorganisms according to the invention.
  • the microorganisms can also be coated with these substances by conjugation.
  • the methods of conjugation are clearly summarized in Sedlacek et al Contributions to Oncology 32: 1-132, 1988.
  • the coating by adsorption takes place, for example, by suspending the microorganisms in a solution preferably containing 0.1 to 50% of the coating substances over a period of preferably 10 minutes to 24 hours and a temperature of preferably 4 degrees Celsius.
  • bacteria whose virulence has been reduced are preferably used as microorganisms.
  • Bacteria are furthermore preferably selected from a group comprising Escherichia coli, Salmonella enterica, Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes, Shigella.
  • Microorganisms in the context of the invention are also membrane casings, so-called ghosts, of living or existing microorganisms.
  • Such membrane casings are manufactured, for example, according to EPA 0540525.
  • the invention relates to pharmaceutical preparations containing the microorganisms according to the invention and the use of this pharmaceutical preparation for the prophylaxis and / or therapy of a proliferative disease.
  • a proliferative disease in the sense of this invention is a disease with excessive or uncontrollable cell proliferation, for example a tumor disease such as carcinoma or sarcoma, leukemia, chronic inflammation, an autoimmune disease or the rejection of an organ transplant.
  • Germination administered to a patient locally or systemically is administered to a patient locally or systemically.
  • the term encased means that on the outside of the membrane of the microorganism, a plurality of the same or different molecules (expressed and / or secreted according to one or more of the features I) to III)) as described above, the geometric degree of coverage being between 0.001 and 1, in particular between 0.01 and 1, for example, can be between 0.1 and 1.
  • the geometric degree of coverage can be calculated from the quotient of the total area of all molecules, in a radial (based on a center of the microorganism) projection into the surface of the microorganism, and the surface of the microorganism. As a rule, a spherical surface of the microorganism is assumed for simplification and is calculated from the volume of the microorganism.
  • the "encased" feature may be optional.
  • Example 1 Construction of a bacterial strain for membrane-based expression of human albumin and beta-glucuronidase
  • Membrane expression can occur in Salmonella by fusing the protein to the C-terminus of the HlyA secretion protein in the presence of the HlyB protein, but in the absence of a fully functional HlyD protein.
  • HlyD must not be completely absent, since otherwise there is no connection between the secretion machine and the TolC protein of the outer membrane (Spreng et al., Mol. Microbiol. 31: 1596-1598, 1999).
  • These examples show one of the possible modifications of the HlyD protein for membrane-based expression.
  • the vector pMOhly DD is constructed, in which no functional HlyD protein is generated.
  • part of the hlyD gene is removed from the vector pMOhlyl by the endonucleases Dralll and Apal. After restriction digestion, the ends are digested with 3 '- 5' exonuclease and the 10923 bps fragment is relegated. The beta-glucuronidase gene is then cloned in-frame to the hlyA gene in this vector.
  • the cDNA is from bglu: amplified from a cDNA library using the following primers by polyvinyl lymerasekettenre force (PCR) (GenBank Accession (Gb) M15182): bglu 5 ': ATGCATTGCAGGGCGGGATGCTGTACC bglu 3': ATGCA ⁇ AAGTAAACGGGCTGTTTTCCAAAC
  • PCR polyvinyl lymerasekettenre
  • Oligonucleotide sequences are shown here, v / ie also in the following 5 '- 3').
  • the primers are chosen so that the gene is amplified without the signal sequence.
  • the product (1899 bps) is subcloned with a suitable "PCR Clonmg Kit", and then the ⁇ 1890 bps fragment is extracted via Nsil digestion.
  • the Nsil fragment is then cloned into the Nsil-cut vector pMOhly DD. This results in the vector pMO DDbglu (Fig. 1). (If the Nsil fragment is cloned into the Nsil-cut vector pMOhlyl, the plasmid pMO bglu is formed, which enables the fusion protein to be secreted).
  • the integration vector for the chromosomal integration of the Albumm-HlyA fusion is created.
  • the vector pMOhly alb is produced.
  • This vector based on pMOhlyl carries a fusion of the albumin cDNA with the HlyA gene.
  • the cDNA of the albumin gene (Gb: A06977) is amplified from a commercially available cDNA bank using PCR and the following Nsil-generating primers:
  • the 1830 bps fragment is subcloned and then cut with Nsil.
  • the 1824 bps fragment is now ligated to pMohlyl digested in Nsil.
  • the finished plasmid pMOhly alb thus expresses HlyB, HlyD and a fusion protein from albumin and HlyA.
  • the Nsil fragment can alternatively also be used in the vector pMO DD, this vector bears the name pMO DDalb.
  • a modification of the cloning strategy already described is used for integration in the Salmonella chromosome (Miller and Mekalanos, J Bacteriol 170: 2575-2583, 1988).
  • the Salmonella aroA gene was first cloned into the vector pUC18 (PCR with the following primers:
  • Primer 5 ' ATGGAATCCCTGACGTTACAACCC
  • Primer 3' GGCAGGCGTACTCATTCGCGC "Blunt” cloning of the 1281 bps fragment into the HincII interface of pUC18). Subsequently, a 341 bps fragment in aroA was removed by HincII digestion and subsequent religion. This vector was named pUC18 aroA 1 . The alb-hlyA fusion gene is then cloned into the vector pUCl ⁇ aroA 'together with the promoter sequence located on pMOhly.
  • the vector pMOhly alb is digested with AacII and Swal and then treated with a 3 '-5' exonuclease.
  • the 3506 bps blunt fragment is extracted and ligated into HincII digested pUCl ⁇ aroA '.
  • the aroA flanked alb-hlyA fragment with the entire activator sequences from the vector pUCaro-alb is cloned into the vector pGP704.
  • pUC aro-alb is digested with Hindlll and then with 5 '-3'
  • Exonuclease treated (blunt). Then digested with EcoRI and the 4497 bps fragment is ligated into the EcoRI / EcoRV (Blunt) digested vector pGP704 (EcoRI / RV fragment: 6387 bps).
  • the integration vector pGParo-alb results (FIG. 2).
  • the vector is transformed into the E.coli strain SMIOlpir. This strain enables the vector to replicate because it forms the p protein required for replication. The vector is then transferred via conjugation into the Salmonella typhi Ty21a acceptor strain, which does not allow replication of the vector. Therefore, only bacteria that have integrated the vector chromosomally are selected by tetracycline selection.
  • the cytoplasmic albumin production is checked by Western blot analysis of the bacterial lysate.
  • St21-alb expresses the alb-hlyA fusion, it can neither secrete it nor express it in the membrane.
  • a plasmid with functional HlyB such as pMO DDbglu
  • HlyD such as pMO bglu
  • the plasmid pMO DDbglu with the strain St21-alb is used.
  • St21-alb pMO DDbglu which uses the Hly secretion system to express both human albumin and human beta-glucuronidase at the membrane. This strain can then be used for pro-drug conversion within the meaning of the patent.
  • Example 2 Construction of a bacterial strain coated with albumin-HlyA fusion, to provide the genetic information of human beta-glucuronidase.
  • the bacterial strain shown in this example is to use passive targeting for DNA encoding human beta-glucuronidase to deliver to tumor cells, which are then to be expressed in the tumor cells.
  • a slightly modified strain as in Example 1 is used for the membrane expression of albumin in this example.
  • Both the gene coding for albumin HlyA and the information for HlyB are to be integrated chromosomally. This strain expresses constitutive me - branch albumin.
  • the vector pMOhly alb described above is digested by BsrBI and EcoRI and then treated with 5 '-3' exonuclease.
  • This digest produces a 5815 bps fragment with blunt ends, which contains the complete prokaryotic activation sequence and the genes hlyC, alb-hlyA and hlyB, but not hlyD.
  • This fragment can now be inserted into the HincII interface of the vector pUCl ⁇ aroA 'described above. This results in the vector pUCaro-alb-B.
  • the 6548 bps fragment can be inserted again into the EcoRI-EcoRV digested vector pGP704 by means of an EcoRI-Nrul digest (FIG. 3).
  • the further procedure (replication and integration in S. typhi Ty21a) then corresponds to the strategy described above.
  • the resulting strain St21-alb-B constitutively expresses membrane-bound albumin-HlyA fusion protein. If a vector encoding HlyD is transfected, the albumin-HlyA fusion protein is secreted.
  • the plasmid for the delivery of the DNA encoding beta-glucuronidase is based on the commercial vector pCMVbeta (Clontech). A fusion of the bglu gene with a secretion signal must first be used for the construction. In this example, the signal peptide of the tPA precursor molecule is to be used.
  • the 5 'UTR of the tPA cDNA (Gb E02027) is amplified by PCR with the following primers using the following primers until the end of the region coding for the signal peptide (amplification with "blunt" -generating polymerase):
  • the resulting plasmid pCMVtp (3972 bps) can now be used for expression of heterologous fusion proteins.
  • the bsp fragment of the bglu (Gb M15182) gene (without sequence for signal peptide) from a suitable cDNA bank is amplified with the following Spei-generating primers:
  • the 1899 bps fragment was ligated into the SpeI digested vector pCMVtp.
  • the resulting plasmid pCMVtp bglu now codes for an N-terminal fusion of the tPA signal peptide with the region of the mature protein of beta-glucuronidase.
  • the plasmid pCMVtp bglu (FIG. 4) is transformed into the strain St21-alb-B. This strain now allows the DNA to be delivered to the tumor tissue with the aid of passive targeting and the expression of the DNA by transfected tumor cells then allows a conversion of suitable pro-drugs.
  • Example 3 Construction of a strain coated with albumin-TolC fusion with membrane-like expression of the extracellular domain of FAS and delivery of a ProDrug converting enzyme
  • the strain shown in this example combines the properties shown in Example 2 with a targeted targeting of (tumor) cells that express Fas ligand (FasL).
  • FasL FasL-expressing tumor cells
  • this strain it is possible to specifically target FasL-expressing tumor cells, such as in certain breast tumors (Herrnring et al., Histochem Cell Biol 113: 189-194, 2000). FasL Expression through Tumor cells have been postulated as a potential mechanism for immune escape, since these cells can eliminate actively attacking, expressing Fas, lymphocytes (Muschen et al., J Mol Med 78: 312-325, 2000).
  • these tumor cells which are very problematic for therapy, can be targeted and then eliminated using an apoptosis-independent mechanism.
  • the carrier strain in this example is based on a fusion of albumin with the TolC protein from E. coli. In this way, a membrane-specific expression of albumin is achieved.
  • the membrane-specific expression of the extracellular domain of Fas takes place via the plasmid pMOhlyDD and the plasmid pCMV-bglu described above is used for delivery.
  • the first step involves generation of the carrier strain expressing TolC albumin.
  • the gene for the fusion protein is first generated, and then this gene is integrated into the Salmonella genome via successive cloning in pUCaroA 'and pGP704, in accordance with the examples above.
  • the TolC gene for E is based on a fusion of albumin with the TolC protein from E. coli. In this way, a membrane-specific expression of albumin is achieved.
  • the membrane-specific expression of the extracellular domain of Fas takes place via the plasmid pMOhlyDD and the plasmid pCMV-
  • coli together with the natural promoter, is present in the plasmid pBRtolC.
  • the 1701 bps fragment was inversely ligated into the Sall cleavage site of the vector pBR322 (Gb J01749), whereby the tet gene was interrupted. Due to the known crystal structure of ' TolC (Koronakis et al., Nature 405: 914-919., 2000) the introduction of heterologous DNA into the singular Kpnl interface in the tolC gene allows the expression of the encoded heterologous fusion protein in an extracellular loop on the Outer membrane. To express albumin, the albumin gene from the cDNA (Gb A06977) is amplified using the following primers generating Kpnl:
  • the DNA can be inserted into the Kpnl-cut vector pBRtolC.
  • the reverse orientation results in the vector pBRtolC-alb.
  • the gene for the tolC-Albu in fusion can now be ligated via EcoRV and PshAI (fragment 3970 bps) in reverse orientation into the HincII site of the vector pUCaroA '.
  • the resulting vector pUCaro-alb-tol (7596 bps) is now linearized with HindIII, 5 '-3' exonuclease is treated and then digested with EcoRI.
  • the 4961 bps fragment is then placed in the
  • the 477 bps fragment is digested with Nsil and inserted into the Nsil digested vector pMOhly DD in frame for the HlyA gene.
  • the resulting vector pMO DD-fas thus produces a membrane-bound Fas fragment after transformation into a Salmonella strain, which can bind to FasL-expressing cells with suitable folding. Consequently these sal onelles can be enriched on FasL-expressing cells, such as tumor cells.
  • the plasmid pCMV bglu (Example 2) is now also transfected into the Salmonella.
  • ProDrug-drug-mediated tumor therapy is possible.
  • the better effectiveness of this example compared to the previous example depends crucially on the correct folding of the extracellular domain of Fas.
  • FasL-specific Fab fragments of monoclonal antibodies (which can be folded correctly in bacteria) can also be used with the same approach as described here. This example shows that with the help of this technique it is possible to construct strains with almost any cell specificity by using suitable specific Fab fragments.

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