Specific antibody-receptor interactions trigger InlAB-independent uptake of listeria monocytogenes into tumor cell lines

<p>Abstract</p> <p>Background</p> <p>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 <it>Listeria monocytogenes </it>strain (Lm-spa⁺), which expresses protein A of <it>Staphylococcus aureus </it>(SPA) and anchors SPA in the correct orientation on the bacterial cell surface.</p> <p>Results</p> <p>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 InlAB-independent 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/receptor-mediated 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.</p> <p>Conclusions</p> <p>Binding of receptor-specific antibodies to SPA-expressing <it>L. monocytogenes </it>may represent a promising approach to target <it>L. monocytogenes </it>to host cells expressing specific receptors triggering internalization.</p

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression

Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice

BACKGROUND: Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models. METHODS: The antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays. RESULTS: C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas. CONCLUSIONS: The combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies

B-Raf specific antibody responses in melanoma patients

Abstract Background Mutations of the BRAF gene are the most common genetic alteration in melanoma. Moreover, BRAF mutations are already present in benign nevi. Being overexpressed and mutated, B-Raf is a potential target for the immune system and as this mutation seems to be an early event, a humoral immune response against this antigen might serve as a diagnostic tool for detection of high risk patients. Methods 372 sera of 148 stage IV melanoma patients and 119 sera of non-melanoma patients were screened for B-Raf, B-Raf V599E and C-Raf specific antibodies by an ELISA assay. Sera were screened for specific total Ig and for IgG. Serum titers were compared with a two tailed Mann-Whitney U test. Sera with titers of 1:300 or higher were termed positive and groups were compared with a two tailed Fisher's exact test. Results B-Raf specific antibodies recognizing both B-Raf and B-Raf V599E were detected in 8.9% of the sera of melanoma patients and in 2,5% of the control group. Raf specific IgG was detected in some patients at very low levels. B-Raf specific antibody responses did not correlate with clinical parameters but in some cases, B-Raf antibodies emerged during disease progression. Conclusion These findings imply that B-Raf is immunogenic in melanoma patients and that it might serve as a potential target for immunotherapy. However, B-Raf specific antibodies emerge at rather late stages of melanoma progression and are present only with a low frequency indicating that spontaneous B-Raf specific antibodies are not an early marker for melanoma, but rather may serve as a therapeutic target.</p