EP1478756A2

EP1478756A2 - Microorganisms as carriers of nucleotide sequences coding for cell antigens used for the treatment of tumors

Info

Publication number: EP1478756A2
Application number: EP03704315A
Authority: EP
Inventors: Ulf R. Rapp; Werner Goebel; Ivaylo Gentschev; Joachim Fensterle
Original assignee: Zentaris AG
Current assignee: Aeterna Zentaris GmbH
Priority date: 2002-02-28
Filing date: 2003-02-13
Publication date: 2004-11-24
Also published as: CN1650014A; PL372370A1; RU2004128929A; IL163672A0; KR20040104464A; NO20043926L; MXPA04008287A; CA2513190A1; AU2003206664A1; DE10208653A1; ZA200407528B; RU2319741C2; JP2005518795A; WO2003072789A3; BRPI0308119A2; US20060105423A1; WO2003072789A2; NZ535312A; RS75604A; HRP20040785A2

Abstract

The invention relates to a microorganism with a nucleotide sequence coding for a cell antigen in which the following components are inserted and are expressible: I) a nucleotide sequence coding for at least one epitope of an antigen of a tumor cell and/or a nucleotide sequence for at least one epitope of an antigen that is specific for a tissue cell from which the tumor originates; II) an optional nucleotide sequence coding for a protein that stimulates cells of the immune system; IIIA) a nucleotide sequence for a transport system which makes it possible to express the expression product of components I) and, optionally, II) on the outer surface of the bacterium and/or secrete the expression product of component I) and, optionally, of component II); and/or IIIB) a nucleotide sequence for a protein used for lysing the microorganisms in the cytosol of mammalian cells and for intracellularly releasing plasmids which are contained in the lysed microorganisms; and IV) an activation sequence for expressing one or several of components I) to IIIB), said activation sequence being selected among the group consisting of an activation sequence which is capable of being activated in the microorganism, is tissue-cell-specific but not cell-specific . Each of components I) to IV) can be identically or differently arranged in an individual or multiple manner. Also disclosed are uses of such a microorganism for the production of a medicament.

Description

Microorganisms as carriers of nucleotide sequences coding for cell antigens for the treatment of tumors.

Field of the Invention.

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.

Background of the Invention and Prior Art.

The main cause of the mostly fatal course of malignant tumor diseases is the inability of the body's defense system to recognize and destroy malignant cancer cells. Cancer is one of the most common fatalities in industrialized countries. In Germany alone, more than 210,000 people die each year from malignant neoplasms (source: WHO, figures from 1997), which corresponds to an annual rate of over 255 deaths per 100,000 inhabitants.

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, 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.

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. In tumor patients, the naturally existing populations of CD8 +, cytotoxic T cells (CTL), are obviously not sufficient to recognize and eliminate the tumor cells (Jaffee, Ann.NYAcad. Sei. 886: 67-72, 1999) , In addition, 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).

It is already known that attenuated intracellular bacteria are suitable as vaccine carriers against certain bacterial infections, which are mainly caused by such mentioned Thl immune response can be combated (Hess and Kaufmann, FEMS Immunology & Medical Microbiology 23: 165-173, 1999). This answer is characterized by CTL and the presence of specific IFN-g secreting 5 CD4 + T cells (also T helper cells, Th) (Abbas ■ et al., Nature 383: 787-793, 1996). Other groups have shown that recombinant bacteria can protect against a heterologous tumor (Medina et al., Eur J Immunol 29: 693-699., 1999; Pan et al., Cancer Res 59: 5264-5269., ¹⁰ 1999; Woodlock et al., J Immunother 22: 251-259., 1999; Paglia et al., Blood 92: 3172-3176., 1998; Paglia et al., Eur J Immunol 27: 1570-1575., 1997; Pan et al ., Nat Med 1: 471-477., 1995; Pan et al., Cancer Res 55: 4776-4779.,

1995). In these cases, however, animals were opposed to one

15

Immunized surrogate antigen, and then applied tumor cells that express this antigen.

However, these tumor systems cannot be compared with clinical tumors, since there was no tolerance for the tumor antigen in these models. 0

A good number of different tumor vaccines have already been clinically tested. However, no breakthrough in the treatment of tumor diseases has yet been achieved with any tumor vaccine or vaccination method. Against this background, there is still an extremely great need for new tumor therapy methods.

It is known to express expression products of nucleic acid sequences introduced into bacteria on the cell membrane of these bacteria or to have them secreted by these bacteria. Basis of this technique is the Eb 0 cherichia coli hemolysin system hlyAs which type the Proto ^¬ of a type I secretion system is of gram negative bacteria. With the help of the HlyAs, secretion vectors were developed that are efficient Allowing protein antigens to be discharged into Salmonella enterica, Yersinia enterocol tica and Vibrio cholerae. Such secretion vectors contain the cDNA of any protein antigen coupled to the nucleotide sequence for the HlyA signal peptide for which hamolysin

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 a much stronger immune protection than vaccines 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; Vacine 19: 2621-2618, 2001, Hess et al PNAS 93: 1458-1463, 1996). However, 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.

A technique for introducing plasmid DNA into sucker cells by carrier bacteria such as Salmonella and Listeria monocytogenes has been developed. Genes contained in these plasmids could also be expressed in the sucker cells when 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. By introducing the nucleotide sequences for a specific lysis gene, the Listeria monocytogenes germs were dissolved in the cytosol of the antigen-presenting cell and released their plasmids, which led to the subsequent expression, processing and presentation of the plasmid-encoded proteins and the immunogenicity of these proteins increased significantly (Dietrich et al. Nat. Biotechnol 16: 181-185, 1998; Vaccine 19: 2506-2512, 2001).

Virulence-attenuated variants of bacteria that colonize intracellularly have been developed. For example, 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. For example, 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. 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. On the other hand, after release from the phagosome, 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. It was also possible to show that the interaction of the listeria with macrophages, natural killer cells (NK) and neutrophil granulocytes resulted in the expression of such cytokines (TNF-alpha, IFN-gamma, 11-2, IL-12; Unanue, Curr. Opin Immunol, 9: 35-43, 1997; Mata and Paterson, J Immunol 163: 1449-14456, 1999) is induced for which antitumor activity has been demonstrated. The administration of L. monocytogenes, which had been transduced to express tumor antigens, inhibited the growth of experimental tumors in an antigen-specific manner (Pan et al Nat Med 1: 471-477, 1995, Cancer Res 59: 5264-5269, 1999; Voest et al. Natl Cancer Inst 87: 581-586, 1995; Beatty and Paterson, J Immunol 165: 5502-5508, 2000).

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:

3440-34450, 2001; Xiang et al. , PNAS 97: 5492-5497, 2000). Recombinant Salmonella strains were also effective as a prophylactic vaccine against viral infections (HPV) (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).

Technical problem of the invention.

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.

Basic concept of the invention. To solve this technical problem, 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 cytosol of suction cells and for the intracellular release of plasmids contain in the lysed microorganisms, and IV) an activation sequence for the expression of one or more of the components I) to IIIB) selected from the group consisting of "tissue cell-specific and non-cell-specific activation sequence that can be activated in the microorganism", where each of the components I) to IV) can be set up one or more times, in each case the same or different, and uses of such a microorganism for producing a medicament.

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. In the end, the invention triggers an immune reaction directed against the tumor.

In detail, 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 the microorganism or is cell-specific, tumor cell-specific, tissue cell-specific or function-specific, for the expression of component I) and II).

Preferred embodiments

The components of a microorganism according to the invention are described in detail below. Component I)

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. Furthermore, 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.

Examples of cell antigens and their oncogenic mutations according to the invention 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, - B, - H, Cdk-1, -2, -4, -6, -7, Cdc25C, P16, pl5, p21, p27, pl8, pRb, pl07, pl30, E2F (1-5), GAAD45, MDM2, PCNA, ARF, PTEN, APC, BRCA, P53 and homologues, IV) transcription factors and their oncogenic mutations such as, for example C-Myc, NFkB, c-Jun, ATF-2, Spl, v) embryonic proteins, such as, for example, carcmoembryonic antigen, alpha-fetoprotem, Mage, PSCA, vi) differentiation antigens, such as, for example, Mart, GplOO, tyrosmase, GRP, TCF-4, vn) viral antigens such as the following viruses: HPV, HCV, HPV, EBV, CMV, HSV. Alternatively or additionally, 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. Component II)

Component II) represents at least one nucleotide sequence for at least one protein which stimulates cells of the immune system. By choosing the protein, 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)

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.

Component IIIA)

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

(Nucleotide sequences containing HlyA, HlyB and HlyD under the control of the hly-specific promoter); The following transport signals are to be used: for secretion - the C-terminal HlyA transport signal, in the presence of HlyB and HlyD proteins; for membrane-based expression - the C-terminal HlyA transport signal, in the presence of the HlyB protein, ii) the hemolysin transport signal from E. coli (nucleotide sequences containing HlyA, HlyB and HlyD under the control of a non-hly-specific bacterial promoter), iii) the transport signal for the S-layer protein (Rsa A) from Caulobacter crescentus; the following transport signals are to be used: for secretion and for membrane-based expression - the C-terminal RsaA transport signal, iv) the transport signal for the TolC protein from Escherichia coli; The following transport signals are to be used: for membrane-based expression - the N-terminal transport signal of TolC (the integral membrane protein TolC from E. coli is a multifunctional, pore-forming protein of the outer membrane of E. coli, which besides functions such as the uptake of Colicin El (Morona et al., J Bacteriol 153: 693-699., 1983) and the secretion of Colicin V (Fath et al., J Bacteriol 173: 7549-7556., 1991) also as Serves as a receptor for the U3 phage (Austin et al., J Bacteriol 172: 5312-5325., 1990); this protein is found not only in E. coli but in a large number of Gram-negative bacteria (Wiener, Structure Fold Des 8: R171-175., 2000); the localization in the outer membrane and the widespread occurrence make TolC an ideal candidate to present heterologous antigens, for example to trigger an immune response).

Component IIIB)

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. Such lytic proteins (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)

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. Such 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.,

Nat.Biotechnol. 16: 181-185, 1998) or the pagC promoter from S. typhimurium (Bumann, Infect Immun 69: 7493-7500., 2001). If the plasmids are released by the microorganism into the cytosol of the cell after its lysis, 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.

In a particular embodiment of this invention, 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 ^Λ 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. After administration of the microorganisms according to the invention, the tolerance for a cell presenting component I), for example for a tumor cell, or for a tissue cell from which the tumor originates, is broken and one directed against the tumor and / or against its tissue cells cytotoxic immune response triggered. Depending on the choice of component I), 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. Among the proliferative diseases are tumor diseases, Leukemia, viral diseases, chronic inflammation, rejection of transplanted organs and autoimmune diseases count.

In a particular embodiment of this invention, in which 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, the pharmaceutical preparation according to the invention for the prophylaxis or therapy of a tumor of the thyroid gland, the breast, the stomach, kidney, ovary, birthmarks, prostate, cervix or bladder.

The invention is explained in more detail below on the basis of examples which merely illustrate embodiments.

Example 1: Induction of an immune response in BxB mice by immunization with Ξalmonella expressing c-Raf

Raf is usually a cytosolic

Serine / threonine kinase (PSK) which, in interaction with other proteins in signal cascades, controls cell growth and survival (Kerkhoff and Rapp, Oncogene 17: 1457-1462., 1998; Troppmair and Rapp, Recent Results Cancer Res 143: 245 -249., 1997). A bond of a

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).

For the test in the mouse model, transgenic mice are used 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.

To generate the vaccine, 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). In immunoblotting using antibodies directed against c-Raf, 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 χo ^A 9), the vaccination being repeated twice with an interval of 5 days. An intravenous inoculation with 5xlO ^Λ 5 salmonellae was given 45 days after the last immunization. As a control, naked c-Raf encoding DNA was administered intramuscularly to mice. 5-7 days after the last immunization, serum samples were now taken and the antibody response was analyzed using an WESTERN blot. For this purpose, the 1: 200 diluted serum against membranes with separated protein was used and blotted protein from c-Raf transfected or non-transfected bacteria. The detection of the bound serum antibodies was carried out with the help of antibodies specific for mouse IgG. In contrast to the control mice, specific antibodies of the IgG isotype could be induced in 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.

To analyze the CD8 + T cell response, 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.

To demonstrate the tumoricidal activity, 10, 12 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. In the group immunized with SL-pMO-Raf, 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). These data thus show that in this experiment a vaccination with SL-pMO-Raf could protect animals from the development of tumors and that the invention described here is suitable as a tumor vaccine. These experiments further show that the carrier system shown in this invention can in principle break self-tolerance and induce a c-Raf specific antibody response and T cell response in animals tolerant of c-Raf. Using the same experimental system, 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.

The existence of 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). During PSA already is already overexpressed in early tumor forms (Watt et al., Proc Natl Acad Sei USA 83: 3166-3170., 1986; Wang et al., Prostate 2: 89-96., 1981) and thus contributes to carcinoma diagnosis (Labrie et al ., J Urol 147: 846-851; discussion 851-842., 1992), is the PSCA-

Expression usually increased only in the locally advanced, undifferentiated and metastatic tumor stage (Gu et al., Oncogene 19: 1288-1296., 2000; Reiter et al., Proc Natl Acad Sei U S A 95: 1735-1740., 1998). The organ specificity makes both PSA and PSCA a potential target antigen in the development of immunotherapies against prostate cancer (Reiter et al., Proc Natl Acad Sei USA 95: 1735-1740., 1998; Hodge et al., Int J Cancer 63:

231-237., 1995; Armbruster, Clin Chem 39: 181-195., 1993).

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. For this purpose, 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. For the

A 645 base pair (bp) fragment was selected for amplification. 5 ^' -GTGGATTG- served as primer

GTGATGCATCCCTCATC-3 'and 5' -CAGGGCACATGCATCACTGCCCCA-3 ^' .

The PCR product was first cloned "blunt-end" into the vector pUClδ 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).

With this Salmonella strain BALB / c mice are now nasally immunized three times at 3 week intervals with a dose of 1x107. The immune response is checked with WESTERN blot

Analyzes and intracellular cytokine staining demonstrated.

Claims

claims

1) Microorganism with a nucleotide sequence coding for a cell antigen, in the genome of which the following components are inserted and can be expressed:

I) a nucleotide sequence coding for at least one epitope of an antigen or more antigens of a tumor cell and / or a nucleotide sequence for at least one epitope of an antigen or more antigens specific for a tissue cell from which the tumor originates,

II) optionally, a nucleotide sequence coding for a protein which stimulates cells of the immune system,

IIIA) a nucleotide sequence for a transport system which enables expression of the expression product of component I) and, optionally, II) on the outer surface of the bacterium and / or secretion of the expression product of component I) and, optionally, component II) , and / or IIIB) a nucleotide sequence for a protein for the lysis of the microorganisms in the cytosol of mammalian cells and for the intracellular release of plasmids contained in the lysed microorganisms, and IV) an activation sequence for the expression of one or more of the components I) to III3) selected from the group consisting of "tissue cell-specific and non-cell-specific activation sequence that can be activated in the microorganism", wherein each of the components I) to IV) can be set up one or more times, in each case the same or different.

2) microorganism according to claim 1), wherein the microorganism em virus, a bacterium, in particular a gram-positive or gram-negative bacterium, preferably selected from the group consisting of "Escherichia coli, Salmonella, Yers ia enterocolitica, Vibrio cholerae, Listeria monocytogenes, and Shigella ", or a unicellular parasite, the virulence of the microorganism preferably reducing

3) Microorganism according to claim 1), wherein the microorganism is the shell of a bacterium.

4) Microorganism according to one of claims 1) to 3), wherein component I) is a nucleotide sequence which codes for a epitope or several epitopes of an antigen or several antigens of a protein or several proteins, optionally mutant, of a tumor cell, this protein is preferably selected from the group consisting of "extracellular, transmembrane or intracellular part of a receptor; extracellular, transmembrane or intracellular part of an adhesion molecule; signal-transducing protein; a cell division-controlling protein; transcription factor; differentiation protein; embryonic protein; and vibratory protein; and ", the protein preferably being an oncogene gene product or a suppressor gene product, in particular c-Raf, A-Raf, B-Raf or a homologous protein from c-Raf, A-Raf or B-Raf.

5) Microorganism according to one of claims 1) to 3), wherein component I) is a nucleotide sequence which codes for an antigen which is specific for the tissue cell, in particular selected from the group consisting of "thyroid, breast, salivary, lymph gland , Mammary gland, gastric mucosa, kidney, ovary, prostate, cervix, bladder mucosa and birthmark ", from which the tumor originates.

6) Microorganism according to one of claims 1) to 5), with a component I) according to claim 4) and one

Component I) according to claim 5).

7) Microorganism according to one of claims 1) to 6), wherein component II) codes for at least one cytokine, interleukin, interferon and / or chemokine.

8) Microorganism according to one of claims 1) to 7), wherein the component IIIA) codes for the hemolysin transport signal from Escherichia coli, for the S-layer (Rsa A) protein from Caulobacter crescentus, or for the TolC protein from Escherichia coli.

9) Microorganism according to one of claims 1) to 8), wherein component IIIB) codes for a lytic protein from gram-positive bacteria, for a lytic protein from Listeria monocytogenes, for PLY551 from Listeria monocytogenes and / or for the Holin from Listeria monocytogenes. 10) Microorganism according to one of claims 1) to 9), wherein component IV) codes for an activator sequence that can be activated in the microorganism, in particular codes for a tumor cell-specific, tissue cell-specific, macrophage-specific, dendrite-specific, lyphocyte-specific, function-specific or non-cell-specific activator sequence ,

10

11) Microorganism according to one of claims 1) to 10), in which component I) codes for at least two different proteins.

15 12) Use of a microorganism according to one of claims 1) to 11) for the manufacture of a medicament, in particular for the prophylaxis and / or therapy of a disease which is caused by uncontrolled cell division or an infection.

20, preferably a tumor disease, in particular a prostate carcinoma, an ovarian carcinoma, a breast carcinoma, a gastric carcinoma, a kidney tumor, a thyroid tumor, a melanoma, a cervical tumor, a bladder tumor,

25 a salivary gland tumor or a lymph gland tumor, a leukemia, a viral or bacterial infection, a chronic inflammation, an organ disorder and / or an autoimmune disease.

30 13) Use according to claim 12) for the removal of a tumor as well as the healthy tissue from which the tumor originates. 14) Use according to claim 12) or 13), wherein the medicament is prepared for local, ^■ - parenteral, oral or rectal administration.

15) Process for the preparation of a medicament according to one of claims 12) to 15), wherein a microorganism according to one of claims 1) to 11) in a physiologically effective dose with one or more physiologically tolerable carriers for oral, im, iV, ip, rectal or local gift is prepared.

16) Plasmid or expression vector containing components I) to IV) according to claim 1.

17) Method for producing an organism according to one of claims 1) to 11), wherein a plasmid or expression vector according to claim 16) is generated and a microorganism is transformed with this plasmid or expression vector.