Non-pathogenic and/or attenuated bacteria capable of inducing apoptosis in macrophages, process of manufacturing and uses thereof

Abstract

The invention relates to an non-pathogenic and/or attenuated bacterium which is capable of inducing apoptosis in macrophages.

1 . A non-pathogenic and/or attenuated bacterium which is capable of inducing apoptosis in macrophages. 
     
     
         2 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the bacterium is capable of infecting macrophages. 
     
     
         3 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the bacterium is selected from the group consisting of gram-negative bacterium and gram-positive bacterium. 
     
     
         4 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the bacterium is selected from the group consisting of  Shigella  spp.,  Salmonella  spp.,  Listeria  spp.,  Mycobacterium  spp.,  Escherichia  spp.,  Yersinia  spp.,  Vibrio  spp., and  Pseudomonas  spp. 
     
     
         5 . The non-pathogenic and/or attenuated bacterium according to  claim 4 , wherein the bacterium is selected from the group consisting of  Shigella flexneri, Salmonella typhimurium, Mycobacterium bovis  BCG,  Listeria monocytogenes, Escherichia coli, Salmonella typhi, Yersinia enterocolitica , and  Vibrio cholerae.    
     
     
         6 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the attenuation is caused by deletion or inactivation of at least one gene selected from the group consisting of aroA, aro, asd, gal, pur, cya, crp, phoP/Q, and omp. 
     
     
         7 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the attenuation results in an auxotrophic bacterium. 
     
     
         8 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the macrophages are M1 macrophages and/or M2 macrophages. 
     
     
         9 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the macrophages are M2 macrophages. 
     
     
         10 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the induction of apoptosis is achieved by caspase activation. 
     
     
         11 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the induction of apoptosis is achieved by caspase-1 activation. 
     
     
         12 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , where the bacterium is a recombinant bacterium. 
     
     
         13 . The non-pathogenic and/or attenuated bacterium according to  claim 12 , wherein the bacterium comprises at least one chromosomally integrated DNA encoding at least one protein selected from the group of: IpaB, and SipB. 
     
     
         14 . The non-pathogenic and/or attenuated bacterium according to  claim 13 , wherein the chromosomally integrated DNA is recombinant DNA, 
     
     
         15 . The non-pathogenic and/or attenuated bacterium according to  claim 13 , wherein the at least one protein is constitutively expressed. 
     
     
         16 . The non-pathogenic and/or attenuated bacterium according to  claim 13 , comprising at least one chromosomal deletion or inactivation of at least one regulatory DNA leading to the constitutive expression of at least one protein. 
     
     
         17 . The non-pathogenic and/or attenuated bacterium according to  claim 13 , wherein the bacterium comprises at least one plasmid. 
     
     
         18 . The non-pathogenic and/or attenuated bacterium according to  claim 17 , wherein the at least one plasmid encodes at least one protein selected from the group of: IpaB and SipB. 
     
     
         19 . The non-pathogenic and/or attenuated bacterium according to  17 , wherein the at least one plasmid encodes at least one regulatory DNA leading to the constitutive expression of the at least one protein. 
     
     
         20 . The non-pathogenic and/or attenuated bacterium according to  claim 1 , wherein the non-pathogenic and/or attenuated bacterium is selected from the group consisting of:  Shigella flexneri  M90T delta-aroA,  Salmonella typhimurium  delta-aroA, and  Shigella flexneri  BS176 delta-aroA pWR100. 
     
     
         21 . A pharmaceutical composition comprising at least one non-pathogenic and/or attenuated bacterium according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         22 . The pharmaceutical composition according to  claim 21 , comprising at least one lyophilized non-pathogenic and/or attenuated bacterium. 
     
     
         23 . A method for treating a physiological and/or pathophysiological condition selected from the group consisting of diseases involving macrophage inflammations where macrophages are associated with disease onset or disease progression, tumor diseases, uncontrolled cell division, malignant tumors, benign tumors, solid tumors, sarcomas, carcinomas, hyperproliferative disorders, carcinoids, Ewing sarcomas, Kaposi sarcomas, brain tumors, tumors originating from the brain, tumors originating from nervous system, tumors originating from the meninges, gliomas, neuroblastomas, stomach cancer, kidney cancer, kidney cell carcinomas, prostate cancer, prostate carcinomas, connective tissue tumors, soft tissue sarcomas, pancreas tumors, liver tumors, head tumors, neck tumors, oesophageal cancer, thyroid cancer, osteosarcomas, retinoblastomas, thymoma, testicular cancer, lung cancer, bronchial carcinomas, breast cancer, mamma carcinomas, intestinal cancer, colorectal tumors, colon carcinomas, rectum carcinomas, gynecological tumors, ovary tumors/ovarian tumors, uterine cancer, cervical cancer, cervix carcinomas, cancer of body of uterus, corpus carcinomas, endometrial carcinomas, urinary bladder cancer, bladder cancer, skin cancer, basaliomas, spinaliomas, melanomas, intraocular melanomas, leukemia, chronic leukemia, acute leukemia, lymphomas, infection, viral or bacterial infection, influenza, chronic inflammation, organ rejection, autoimmune diseases, diabetes and diabetes type II, the method comprising administering to an individual in need thereof an effective amount of the non-pathogenic and/or attenuated bacterium according to  claim 1 . 
     
     
         24 . The method according to  claim 21 , wherein
 (a) apoptosis is induced in tumor-associated macrophages (TAM) and tumor-associated macrophages (TAM) are partially or completely depleted and/or   (b) apoptosis is induced in disease-associated macrophages and disease associated macrophages are partially or completely depleted.   
     
     
         25 . The method according to  claim 23 , wherein the non-pathogenic and/or attenuated bacterium is administered before and/or during and/or after administering at least one further pharmacologically active substance. 
     
     
         26 . The method according to  claim 25 , wherein the further pharmacologically active substance is selected from the group consisting of: a DNA topoisomerase I inhibitor, a DNA topoisomerase II inhibitor, a DNA intercalator, an alkylating agent, a microtubuli destabilizer, a hormone receptor agonist, a growth factor receptor agonist, a hormone receptor antagonist, a growth factor receptor agonist, an inhibitor of signal transduction, an antibody against a growth factor, an antibody against a growth factor receptor, a kinase inhibitor, and an antimetabolite. 
     
     
         27 . The method according to  claim 25 , wherein the further pharmacologically active substance is selected from the group consisting of actinomycin D, aminoglutethimide, asparaginase, avastin, azathioprine, BCNU (carmustine), bleomycin, busulfan, carboplatin, CCNU (lomustine), chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, diethylstilbestrol, doxorubicin (adriamycin), DTIC (dacarbacin), epirubicin, erbitux, erythrohydroxynonyladenine, ethynyloestradiol, etoposide, fludarabine phosphate, fluoxymesterone, flutamide, gemcitabine, Gleevec/Glivec, Herceptin, hexamethylmelamine, hydroxyurea, hydroxyprogesterone caproate, idarubicin, ifosfamide, interferon, iressa, irinotecan, Lasparaginase, leucovorin, mechlorethamine, riedroxyprogesterone acetate, megestrol acetate, melphalan, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, N-phosphonoacetyl-L-aspartate (PALA), oxaliplatin, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifen, rapamycin, semustine, sorafenib, streptozocin, tamoxifen, tarceva, taxotere, teniposide, testosterone propionate, thioguanine, thiotepa, topotecan, trimethylmelamine, uridine, vinblastine, vincristine, vindesine, vinorelbine, 2′,2′-difluorodeoxycytidine, 5-fluorodeoxyuridine monophosphate, 5-azacytidine cladribine, 5-fluorodeoxyuridine, 5-fluorouarcil (5-FU), and 6-mercaptopurine. 
     
     
         28 . The method according to  claim 25 , wherein the non-pathogenic and/or attenuated bacterium is administered before and/or during and/or after the treatment with radiotherapy and/or surgery. 
     
     
         29 . A process for the production of non-pathogenic and/or attenuated bacterium according to  claim 1 , the method comprising one or more of the following:
 (a) deleting or inactivating at least one gene selected from the group consisting of: aroA, aro, asd, gal, pur, cya, crp, phoP/Q, and omp in a non-pathogenic and/or non-attenuated bacterium;   (b) integrating a DNA into the genome of a non-pathogenic and/or attenuated bacterium, comprising DNA which encodes at least one protein selected from the group of: IpaB, and SipB;   (c) introducing at least one plasmid into the non-pathogenic and/or attenuated bacterium, which comprises DNA which encodes at least one protein selected from the group of: IpaB, and SipB;   (d) integrating a DNA into the genome of the non-pathogenic and/or attenuated bacterium, which comprises at least one regulatory DNA which enables the constitutive expression of at least one protein selected from the group of: IpaB and SipB;   (e) chromosomally deleting or inactivating at least one regulatory DNA which leads to the constitutive expression of at least one protein selected from the group of: IpaB and SipB; and   (f) introducing at least one plasmid into the non-pathogenic and/or attenuated bacterium, which comprises at least one regulatory DNA which enables the constitutive expression of at least one protein selected from the group of IpaB and SipB.   
     
     
         30 . A pharmaceutical kit comprising at least one non-pathogenic and/or attenuated bacterium according to  claim 1  and a pharmacologically acceptable buffer for intravenous injection.