TY - JOUR A1 - Heisig, Martin A1 - Frentzen, Alexa A1 - Bergmann, Birgit A1 - Gentschev, Katharina Ivaylo A1 - Hotz, Christian A1 - Schoen, Christoph A1 - Stritzker, Jochen A1 - Fensterle, Joachim A1 - Rapp, Ulf R. A1 - Goebel, Werner T1 - Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines N2 - Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization. KW - Listeria monocytogenes Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68705 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Josupeit, Rafael A1 - Rudolph, Stephan A1 - Ehrig, Klaas A1 - Donat, Ulrike A1 - Weibel, Stephanie A1 - Chen, Nanhai G. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Heisig, Martin A1 - Thamm, Douglas A1 - Stritzker, Jochen A1 - MacNeill, Amy A1 - Szalay, Aladar A. T1 - Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma JF - PLoS One N2 - Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS. KW - breast-tumors KW - animal-model KW - nude-mice KW - cell-line KW - in-vitro KW - glv-1h68 KW - cancer KW - virotherapy KW - dogs KW - neutrophils Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129998 VL - 7 IS - 5 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Müller, Meike A1 - Adelfinger, Marion A1 - Weibel, Stephanie A1 - Grummt, Friedrich A1 - Zimmermann, Martina A1 - Bitzer, Michael A1 - Heisig, Martin A1 - Zhang, Qian A1 - Yu, Yong A. A1 - Chen, Nanhai G. A1 - Stritzker, Jochen A1 - Lauer, Ulrich M. A1 - Szalay, Aladar A. T1 - Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68 JF - PLOS ONE N2 - Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man. KW - Breast-tumors KW - Nude-mice KW - In-vivo KW - Cancer KW - Inhibitor KW - Tissue KW - Agent KW - COX-2 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135319 VL - 6 IS - 7 ER - TY - THES A1 - Heisig, Martin T1 - Development of novel Listeria monocytogenes strains as therapeutic agents for targeted tumor therapy T1 - Entwicklung von neuen Listeria monocytogenes Stämmen zur Anwendung in der Tumortherapie N2 - Despite marked progress in development and improvement of cancer therapies the rate of cancer related death remained stable over the last years. Especially in treating metastases alternative approaches supporting current therapies are required. Bacterial and viral vectors have been advanced from crude tools into highly sophisticated therapeutic agents detecting and treating neoplastic leasions. They might be potent enough to fill in this therapeutic demand. In this thesis Listeria monocytogenes was investigated as carrier for targeted bacterial cancer therapy. One part of the study focussed on modification of a functional bacterial mRNA delivery system. Genomic integration of T7 RNA polymerase driving mRNA production allowed reduction to an one-plasmid-system and thereby partially relieved the growth retardation exerted by mRNA delivery. Importantly the integration allowed metabolic attenuation of the mRNA delivery mutant potentially enabling in vivo applications. Further expansion of the bacterial RNA delivery system for transfer of shRNAs was examined. Bacterial mutants producing high amounts of RNA containing shRNA sequences were constructed, however a functional proof of gene silencing on delivery in eukaryotic cell lines was not achieved. The second part of this thesis focussed on increasing tumor colonization by Listeria monocytogenes in vivo. Coating bacteria with antibodies against receptors overexpressed on distinct tumor cell lines enabled specific bacterial internalization into these cells in vitro. Optimization of the bacterial antibody coating process resulted in an up to 104-fold increase of intracellular bacteria. Combination of this antibody-mediated targeting with the delivery of prodrug-converting enzymes showed a cytotoxic effect in cell lines treated with the corresponding prodrug. Since incubation in murine serum completely abrogated antibodymediated bacterial internalization the antibodies were covalently linked to the bacteria for application in xenografted tumor mice. Bacteria coated and crosslinked in this manner showed enhanced tumor targeting in a murine tumor model demonstrating antibodymediated bacterial tumor targeting in vivo. Independent of antibody-mediated tumor targeting the intrinsic tumor colonization of different Listeria monocytogenes mutants was examined. Listeria monocytogenes ΔaroA ΔinlGHE colonized murine melanoma xenografts highly efficient, reaching up to 108 CFU per gram of tumor mass 7 days post infection. Taken together the presented data shows highly promising aspects for potential bacterial application in future tumor therapies. Combination of the delivery systems with antibodymediated- and intrinsic bacterial tumor targeting might open novel dimensions utilizing Listeria monocytogenes as therapeutic vector in targeted tumor therapy. N2 - Die Weiterentwicklung der Therapiemöglichkeiten bei Krebserkrankungen hat trotz deutlicher Fortschritte nicht zu einer grundsätzlichen Verringerung der krebsbedingten Sterberate geführt. Besonders in der Behandlung von Metastasen werden alternative Therapieansätze zur Unterstützung der etablierten Standardmethoden benötigt. Insbesondere bakterielle und virale Vektoren wurden von groben Werkzeugen zu hochtechnischen therapeutischen Instrumenten verfeinert und könnten in der Zukunft diese therapeutische Lücke schliessen. In dieser Arbeit wurden verschiedene Aspekte der Anwendung von Listeria monocytogenes in der bakteriellen Tumortherapie untersucht. Im ersten Teil der Arbeit wurde das bereits publizierte System zur bakteriellen Übertragung von mRNAs modifiziert. Die genomische Integration der T7 RNA Polymerase, welche die mRNA herstellt, erlaubte die Reduktion der Plasmidzahl und verringerte die Stoffwechselbelastung auf die RNA-übertragenden Bakterien. Diese Integration erlaubte zum ersten Mal die metabolische Attenuation von RNAübertragenden Listerien und ermöglicht damit den in vivo Einsatz dieser Stämme. Als Erweiterung zur bakteriellen mRNA Übertragung wurde die Übetragung von shRNAs untersucht. Obwohl große Mengen an bakteriellen RNAs nachgewiesen wurden, die die shRNA Sequenz enthielten, konnte nach Infektion eukaryotischer Zellen mit diesen Bakterienmutanten keine funktionale Genregulation nachgewiesen werden. Im zweiten Teil dieser Arbeit wurde mit verschiedenen Methoden die Kolonisierung von Tumoren durch Listeria monocytoges verbessert. Durch Beladung der Bakterien mit Antikörpern gegen Rezeptoren, die auf bestimmten Tumorzellen überexprimiert werden, wurde die bakterielle Internalisierung in diese Zelllinien ermöglicht. Durch Optimierung des Antikörper-Beladungsprozesses konnte die Antikörper-vermittelte bakterielle Internalisierung auf das 104 fache der Kontrollen gesteigert werden. In Verbindung mit der Übertragung von Enzymen, die Medikamentenvorstufen in cytotoxische Medikamente umwandeln, konnten in Zellen, die mit der Medikamentenvorstufe inkubiert wurden, zytotoxische Effekte beobachtet werden. Da aber die Behandlung Antikörper-beladener Bakterien mit murinem Serum die spezifische Internalisierung vollständig verhindert, wurden die Antikörper für die Anwendung in Xenograft Tumormäusen kovalent an die Bakterien gebunden. Auf diese Weise behandelte Bakterien zeigten eine verbesserte Tumorzielsteuerung bei Beladung mit tumorbindenden Antikörpern. Unabhängig von der Antikörper-vermittelten Tumorzielsteuerung wurde die Tumorkolonisation verschiedener Listeria monocytogenes Mutanten untersucht. Insbesondere Listeria monocytogenes ΔaroA ΔinlGHE kolonisierte Melanom-Xenograft Tumoren sehr effizient und erreicht bis zu 108 CFU pro Gramm Tumormasse. Zusammengefasst zeigt diese Arbeit verschiedene vielversprechende Aspekte der bakteriellen Tumortherapie, die möglicherweise in Zukunft angewandt werden können. Durch Verbindung der RNA-Transfersysteme mit Antikörper-vermittelter- und intrinsischer Tumorzielsteuerung können neue Möglichkeiten für den Einsatz von Listeria monocytogenes als therapeutischer Vektor geschaffen werden. KW - Krebs KW - Therapie KW - Angewandte Mikrobiologie KW - bacterial cancer therapy KW - bacterial tumor targeting Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48628 ER -