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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • A61P31/12—Antivirals
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  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/255—Salmonella (G)
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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  • C12N1/00—Microorganisms; 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
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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
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  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
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  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Microorganisms as carriers of nucleotide sequences coding for cell antigens for the treatment of tumors.
• the invention relates to a microorganism with foreign nucleotide sequences, its use as a medicament, in particular vaccum, a plasmid with the foreign nucleotide sequences and a method for producing such a microorganism.
• This invention is based on more recent findings in the molecular mechanisms which lead to malignant new formation. Characteristic changes in the control of cell growth and / or cell differentiation take place very early in the development of cancer (Ponten, Cancer Surv 32: 5-35., 1998). Proteins of signal transduction and cell cycle control, which have been identified in recent years and all of which also represent tumor antigens, are significantly involved in these changes. Tumor antigens are roughly divided into three classes (Pardoll, Nat Med 4: 525-531., 1998): i) Tumor-specific neoantigens which are present in the tumor cell in mutated and / or overexpressed form, such as, for. B.
• EGF-R EGF-R
• HER-2 ii) tumor-specific embryonic antigens, such as representatives of the MAGE protein family or CEA, iii) tumor tissue-specific differentiation antigens, such as tyrosinase, Mart-1 / melan-A and gplOO.
• tumor-specific embryonic antigens such as representatives of the MAGE protein family or CEA
• tumor tissue-specific differentiation antigens such as tyrosinase, Mart-1 / melan-A and gplOO.
• CD8 + T cells Effective induction of CD8 + T cells is of crucial importance for the effectiveness of a tumor vaccine, since tumor cells usually do not present MHC class II molecules and the intracellular tumor antigens are mostly restricted to MHC class I.
• CTL cytotoxic T cells
• tumor-specific T cells often cannot effectively attack the tumor tissue through different mechanisms (anergy, tolerance, neutralization) (Smyth et al., Nat Immunol 2: 293-299., 2001).
• a successful tumor vaccine must therefore break this anergy or tolerance and induce a sufficient number of activated, specific CTL as well as specific antibodies.
• the role of specific antibodies is shown in the successful use of monoclonal antibodies (mAb) against tumor antigens of group (a), such as the already commercially available herceptin, a mAb against HER-2 (Colomer et al., Cancer Invest 19: 49-56., 2001).
• 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. Typhi urium and Typhi, as well as Mycobacterium bovis such variants have already been used as well-tolerated live vaccines against typhoid and tuberculosis.
• These bacteria, including their weakened 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 of antigen-presenting cells (APC; macrophages and dendritic cells), which in turn express the co-stimulating molecules and trigger an efficient stimulation of T cells.
• APC 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.
• the invention is based on the technical problem of specifying a medicament which, particularly in tumor prophylaxis and tumor therapy, represents an improved vaccine with a breakdown of the immune tolerance to tumors.
• the invention teaches a microorganism with a nucleotide sequence coding for a cell antigen, in whose genome the following components are inserted and expressible: I) a nucleotide sequence coding for at least one epitope of an antigen or several antigens of a tumor cell and / or a nucleotide sequence for at least one epitope of an antigen or several antigens specific for a tissue cell from which the tumor originates, II) optional, a nucleotide sequence coding for a protein which stimulates cells of the immune system, IIIA) a nucleotide sequence for a transport system which Expression of the expression product of components I) and, optionally, II) on the outer surface of the bacterium and / or the secretion of the expression product of component I) and, optionally, of component II) and / or IIIB) enables a nucleotide sequence for a protein Lysis of the microorganisms in the
• the invention thus relates to microorganisms which are carriers of nucleotide sequences which code for cell antigens which in turn are expressed or secreted on the outer membrane of the microorganisms and the use of these microorganisms for Breakthrough of immune tolerance to tumors, and new tumor vaccines, which contain microorganisms as carriers of nucleotide sequences coding for cell antigens of normal cells and / or of tumor cells.
• the invention triggers an immune reaction directed against the tumor.
• the microorganisms according to the invention contain the following components: I) at least one nucleotide sequence coding for at least one epitope of at least one antigen of at least one cell protein of a tumor cell and / or optionally at least one nucleotide sequence for at least one epitope of at least one antigen specific for the tissue cell, of which the tumor II) optionally at least one nucleotide sequence for at least one protein which stimulates cells of the immune system, IIIA) at least one nucleotide sequence for a transport system for membrane-based expression or for secretion of the cell antigen coded by component I) and for secretion of the component II) encoded immunostimulating protein, IIIB) optionally a nucleotide sequence for a lysine which lyses the microorganism in the cytosol so that plasmids contained in the microorganism are released into the cytosol, IV) at least one nucleotide sequence for one Activation sequence that can be activated in
• Component I) represents at least one nucleotide sequence for at least one epitope of at least one antigen of at least one cell protein or at least one oncogenic mutated cell protein of a tumor cell.
• the oncogenic mutation of the cell protein may have caused a loss or gain in its original cellular functions.
• this cell protein can be selected from the group consisting of "receptor molecules or parts thereof, namely extracellular, transmembrane or cell-internal parts of the receptors; adhesion molecules or parts thereof, namely extracellular, transmembrane or cell-internal parts of the adhesion molecules; proteins of signal transduction; proteins of the cell cycle control "Differentiation proteins; embryonic proteins; and virus-induced proteins".
• Such cell antigens take over the regulation of cell growth and cell division in the cell and are presented on the cell membrane of normal cells, for example by the MHC class I molecule. These cell antigens are often overexpressed or specifically mutated in tumor cells. Such mutations can result in functional impairments of oncogene suppressors or the activation of proto-oncogenes to oncogenes and all of them or together with overexpression can be significantly involved in tumor growth.
• Such cell antigens are presented on the membrane of tumor cells and accordingly represent antigens on tumor cells, but without triggering an immune reaction that influences the patient's tumor disease.
• Rapp US Pat. No. 5,156,841 has already described the use of oncoproteins, ie expression products of the oncogenes Immunogen for tumor vaccines described. Reference is expressly made to this reference.
• cell antigens and their oncogenic mutations are 1) receptors, such as Her-2 / neu, androgen receptor, estrogen receptor, Midke receptor, EGF receptor, ERBB2, ERBB4, TRAIL receptor, FAS, TNFalpha receptor, n) signal-transducing proteins and their oncogenic mutations such as c-Raf (Raf-1), A-Raf, B-Raf, Ras, Bcl-2, Bcl-X, Bcl-W, Bfl-1 , Brag-1, Mcl-1, AI, Bax, BAD, Bak, Bcl-Xs, Bid, Bik, Hrk, Bcr / abl, Myb, C-Met, IAP1, IA02, XIAP, ML-IAP LIVIN, Surviv, APAF-1; m) proteins of the cell cycle control and their oncogenic mutations such as, for example, Cyclm - D (1-3), - E, - A,
• component I) can represent at least one nucleotide sequence for at least one antigen specific for a normal tissue cell from which the respective tumor is derived.
• Specific antigens of this type are, for example, l) receptors such as, for example, androgen receptors, estrogen receptors, lactoferrm receptor, n) differentiation antigens such as, for example, basic myelin, alpha-lactalbu m, GFAP, PSA, fibrillary acid protein, tyrosmase, EGR-1, MUC1.
• l) receptors such as, for example, androgen receptors, estrogen receptors, lactoferrm receptor
• n) differentiation antigens such as, for example, basic myelin, alpha-lactalbu m, GFAP, PSA, fibrillary acid protein, tyrosmase, EGR-1, MUC1.
• Component II represents at least one nucleotide sequence for at least one protein which stimulates cells of the immune system.
• the immune response to the expression product of component I) can be increased and / or more can be directed to the activation of Thl cells (for the cellular immune reaction) or for the activation of Th2 cells (for the humoral immune reaction).
• Immunostimulating proteins are, for example, l) cytok e such as M-CSF, GM-CSF, G-CSF, n) interferons such as IFN-alpha, -ß, gamma, m) interleukms such as IL-1, -2, -3, -4 , -5, -6, -7, -9, -10, -11, -12, -13, -14, -15, -16, Human Leukemia hibitory factor (LIF), IV)
• cytok e such as M-CSF, GM-CSF, G-CSF, n
• interferons such as IFN-alpha, -ß, gamma
• m) interleukms such as IL-1, -2, -3, -4 , -5, -6, -7, -9, -10, -11, -12, -13, -14, -15, -16, Human Leukemia hibitory factor (LIF), IV
• Chemokme such as Rantes, Monocyte chemotactic and activating factor (MCAF), Macrophage mflammatory protem-1 (MIP-1 alpha, -ß), Neutrophil activatmg Protem-2 (NAP-2), IL-8.
• MCAF Monocyte chemotactic and activating factor
• MIP-1 alpha, -ß Macrophage mflammatory protem-1
• NAP-2 Neutrophil activatmg Protem-2
• IL-8 such as Rantes, Monocyte chemotactic and activating factor (MCAF), Macrophage mflammatory protem-1 (MIP-1 alpha, -ß), Neutrophil activatmg Protem-2 (NAP-2), IL-8.
• Component IIIA) is at least one nucleotide sequence, coding for at least one transport system, which enables expression of the expression products of components I) and, optionally, II) on the outer surface of the microorganism.
• the respective component can either be secreted or on the membrane of the microorganism, i.e. to be expressed at the membrane.
• Transport systems of this type are, for example, l) the Hamolysm transport signal from E. coli
• Component IIIB is a nucleotide sequence coding for at least one lytic protein which is in the cytosol a mammalian cell is expressed and the microorganism lysed 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) and / or the Listeria-specific holin under the control of a listerial promoter.
• a preferred embodiment of this invention is the combination of different components IIIB), for example the combination of a lysis protein with the holin.
• the components IIIA and / or IIIB can be constitutively active.
• Component IV) represents at least one nucleotide sequence for at least one activation sequence for the expression of component I) and, optionally, II). If the expression is in the membrane on the outer surface of the microorganism, the activation sequence should preferably be selected such that it can be activated in the microorganism.
• activation sequences are, for example: i) 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 ), ii) inducible promoters, preferably promoters that become active after being taken up into the cell. These include the actA promoter from L. monocytogenes (Dietrich et al.,
• the activation sequence is non-cell-specific, tissue cell-specific, cell cycle-specific or function-specific. Activation sequences which are particularly activated in macrophages, dendritic cells and lymphocytes are preferably chosen.
• Microorganisms in the sense of this invention are viruses, bacteria or unicellular parasites which are usually used for the transfer of nucleotide sequences foreign to the microorganism.
• the microorganisms are gram-positive or gram-negative bacteria, preferably bacteria such as, for example, Escherichia coli, Salmonella, Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes, Shigella.
• Bacteria which are attenuated in their virulence are preferably used.
• the components according to the invention are introduced into the microorganisms using the methods known to the person skilled in the art. If the microorganisms are bacteria, the components are inserted into plasmids and the plasmids are transferred into the bacteria. The techniques and plasmids suitable for this are well known to the person skilled in the art.
• the invention relates to pharmaceutical preparations containing the microorganisms according to the invention or membrane shells of these microorganisms. These membrane casings are produced, for example, by the method described in patent application EP-A-0 540 525.
• Such pharmaceutical preparations are, for example, suspensions of the microorganisms according to the invention in the solutions familiar to pharmacists, suitable for injection.
• Another object of the invention is the administration of a pharmaceutical preparation containing the microorganisms according to the invention.
• the administration takes place locally or systemically, for example in the epidermis, in the subcutis, in the muscles, in a body cavity, in an organ, in the tumor or in the bloodstream.
• a particular subject of this invention is the oral or rectal administration of the pharmaceutical preparation according to the invention for the prophylaxis and / or therapy of a proliferative disease.
• the administration can take place once or several times. With each administration in the range of 10 to 10 ⁇ 9 microorganisms according to the invention are administered. If the administration of this number to the microorganisms according to the invention does not produce a sufficient immune reaction, the number to be injected should be increased.
• the tolerance for a cell presenting component I is broken and one directed against the tumor and / or against its tissue cells cytotoxic immune response triggered.
• this cytotoxic immune reaction is either directed exclusively against the tumor or also against the tumor cells including the tissue cells from which the tumor cells originate.
• the invention thus relates to the administration of a pharmaceutical preparation according to the invention for the prophylaxis or therapy of a proliferative disease.
• the proliferative diseases are tumor diseases, Leukemia, viral diseases, chronic inflammation, rejection of transplanted organs and autoimmune diseases count.
• component I) represents at least one cell antigen which is expressed by a tumor cell and the tissue cells from which the tumor is derived
• Example 1 Induction of an immune response in BxB mice by immunization with ⁇ almonella expressing c-Raf
• Raf is usually a cytosolic
• PSK Serine / threonine kinase
• Ras Growth factor to a corresponding receptor normally leads to activation of Ras, the subsequent activation of Raf via several phosphorylation steps via the PSK and tyrosine kinase MEK and the PSK ERK to activation of the replication machinery in the cell nucleus (Kerkhoff and Rapp, Oncogene 17: 1457-1462., 1998).
• the first link in this chain, the small G protein Ras is found in approximately 30% of all human beings Tumors changed before (Zachos and Spandidos, Crit Rev Oncol Hematol 26: 65-75., 1997).
• Raf is an effector of Ras and is overexpressed in a variety of human tumors (Naumann et al., Recent Results Cancer Res 143: 237-244., 1997).
• mice which overexpress the entire molecule or the constitutively active casedomane (BxB) (Kerkhoff et al., Cell Growth Differ 11: 185-190., 2000). This means that these mice develop lung tumors spontaneously after about half a year.
• BxB constitutively active casedomane
• the human c-Raf cDNA was cloned into the plasmid pMOhly 1 with the aid of PCR “m-frame” with HlyA (FIG. 1).
• the plasmid pMO-Raf was then transfected into attenuated Salmonella (S.typhi murium SL7207), which have a defect in the aromatic metabolism (Hoiseth and Stocker, Nature 291: 238-239, 1981).
• the c-Raf HlyAs fusion protein could be detected in the bacterial lysate and in the culture supernatant of SL7207 bacteria transfected with pMOhy-Raf.
• BxB transgenic mice were then immunized orally with Salmonella at the age of 7-10 weeks (dose 5 x ⁇ o A 9), the vaccination being repeated twice with an interval of 5 days.
• An intravenous inoculation with 5xlO ⁇ 5 salmonellae was given 45 days after the last immunization.
• naked c-Raf encoding DNA was administered intramuscularly to mice.
• serum samples were now taken and the antibody response was analyzed using an WESTERN blot.
• the 1: 200 diluted serum against membranes with separated protein was used and blotted protein from c-Raf transfected or non-transfected bacteria.
• mice immunized with SL7207 c-Raf transfected with pMohly-Raf This shows that immunization with the described Salmonella can break self-tolerance and induce CD4 + T cells, which are necessary for the antibody to change its isotype to IgG.
• C57BL-6 mice were immunized using the same protocol. Spleen cells were isolated 7 days after the last immunization and stimulated with Raf overexpressing EL-4 cells. 1 h after the start of the stimulation, vesicular transport was blocked by brefeldin A and after a further 4 h the cells were stained with CD8 and IFN-g specific antibodies and analyzed by flow cytometry (Mittrucker et al., Infect Immun 70: 199-203., 2002 ). A Raf-specific antibody response could only be detected in a mouse immunized with pMO-Raf.
• mice 10 and 14 month old immunized and non-immunized BxB mice were sacrificed and the lung mass was weighed.
• the lung mass is a direct measure of the size of the tumor.
• mice with a reduction in lung mass were found significantly more frequently after 14 months than in the control groups, including the group which immunized with naked DNA coding for c-Raf (SL-pCMV-raf) had been.
• Tumor growth in untreated animals is usually irreversible (Kerkhoff et al., Cell Growth Differ 11: 185-190., 2000).
• salmonella can be produced as vaccines, which isoforms of C-Raf (such as B-Raf and A-Raf), mutated C-Raf, B-Raf or A-Raf, epitopes of normal or mutated C- Express Raf, B-Raf or A-Raf, or combinations of epitopes from normal and / or mutant C-Raf, B-Raf or A-Raf.
• Examples of a mutation which is associated with a loss of Raf activity are mutations in the Ras-binding domain, the kinase domain and / or the phosphorylation sites.
• Example 2 Induction of an immune response in BALB / c mice by immunization with Salmonella expressing PSA.
• tissue-specific antigens in particular those that are increasingly synthesized and expressed by tumor cells, forms a possible target for therapeutic approaches in addition to the diagnostic utility of these markers.
• Been for prostate cancer so far are three significant anti ⁇ gene identified: PSA (prostate specific At ⁇ term), PSMA (prostate specific membrane antigen) and PSCA (prostate stem cell antigen).
• the aim of this experiment was to show whether PSA-secreting salmonella based on the vector pMOHLY 1 can induce an immune response in BALB / c mice.
• two Nsil cleavage sites were first introduced into the c-DNA sequence of PSA by means of polymerase chain reaction (PCR) in order to enable an "in-frame" insertion of the amplified fragment into the target vector.
• PCR polymerase chain reaction
• a 645 base pair (bp) fragment was selected for amplification. 5 ' -GTGGATTG- served as primer
• the PCR product was first cloned "blunt-end" into the vector pUCl ⁇ and later ligated to the target vector pMOhlyl via Nsil interfaces. Correct insertion was controlled by restriction digestion and by Sequen ⁇ cation confirmed ( Figure 2).

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