Bacteria-mediated tumor therapy has been investigated for over a century . The ability of bacteria to colonize malignant tissue has been exploited in different therapeutic approaches [2, 3].
The delivery of therapeutic agents by bacteria to the tumor represents a promising approach to eradicate the tumor from the inside [4, 5]. A major prerequisite is the specific bacterial colonization of tumor tissue without simultaneous colonization of healthy tissue.
Obligate anaerobic bacteria like Clostridia or Bifidobacteria colonize solely the anoxic parts of tumors due to their inability to tolerate oxygen [6, 7]. For facultative anaerobic bacteria like Salmonella, Escherichia, Vibrio or Listeria, specific tumor colonization has been described and different therapeutic approaches were investigated [4, 8–11]. In general, virulence-attenuated Gram-positive bacterial pathogens, such as Listeria monocytogenes, may be better suited for the systemic application of bacteria in tumor therapy as these bacteria lack the LPS of gram-negative bacteria. LPS may induce strong immune reactions culminating in septic shock after release into the blood stream.
Listeria monocytogenes (Lm) has been successfully studied as carrier for the delivery of DNA and RNA into mammalian cells [12, 13]. In this case pathogenicity of the listerial carrier strain was attenuated by the deletion of aroA . In contrast to most other applied virulence-attenuated Lm strains [10, 15, 16], the aroA mutant possesses all virulence factors, thus enabling the carrier bacteria to invade mammalian cells, escape from the phagosome, and replicate in the cytosol of infected host cells. The intracellular replication rate of the aroA mutant was, however, lower compared to the according wild-type strain and the capability of cell-to-cell spread was drastically reduced .
The cytosolic life cycle of Lm poses an advantage for the delivery of nucleic acids harboring eukaryotic expression cassettes compared to other intracellular bacteria like Salmonella, which reside and replicate in phagosomal compartments. The utilization of Lm as a carrier for the direct delivery of prodrug-converting-enzymes and for the introduction of DNA encoding these enzymes into tumor cells in vitro was successfully assessed recently . Internalization of Lm into non-phagocytic mammalian cells is mainly triggered by the two internalins A and B encoded by the inlAB operon [reviewed in 18]. The deletion of inlAB thus strongly reduces the ability of Lm to actively invade such host cells, but does not change their passive uptake by phagocytic cells.
The targeting of carrier microorganisms to cell surface proteins of specific cells was first performed in viral gene therapy . By genetic fusion of Staphylococcus aureus protein A (SPA) to viral coat proteins monoclonal antibodies recognizing specific receptors on the target cells were fixed to the viral surface. Due to the thereby achieved specific virus/cell interaction, uptake of the viral carrier by the selected target cells could be obtained. Alternatively, single chain antibody fragments (scFv) were expressed on the viral surface which - by the interaction with specific receptors on the host cell surface - led to preferential viral infection of the specific target cells as well.
Many tumor cells overexpress specific marker proteins on their surface which include oncoproteins. HER1 (ErbB1) and HER2 (ErbB2), members of the EGFR/HER family, represent such prominent surface proteins [20, 21]. Enhanced expression or mutational activation of these cell surface proteins leads to tumor progression and generally correlates with poor prognosis in tumor therapy [reviewed in 22]. Both tumor markers, HER1 and HER2, are specifically recognized by the chimeric/humanized monoclonal antibodies, Erbitux (Cetuximab) and Herceptin (Trastuzumab) which are approved for therapy of colorectal carcinoma and breast cancer, respectively.
Antibody-mediated targeting of bacteria to tumor cells was described so far only for Salmonella enterica serovar Thyphimurium expressing a scFv against carcino-embryonic-antigen CEA. Antibody expression resulted in a 2-fold increase of these bacteria in the tumor tissue .
As a novel approach we describe in this study the construction of a virulence-attenuated Lm strain with deletions in inlAB and aroA which expresses functional SPA anchored to the cell wall. This strain, when coated with Herceptin or Erbitux, triggered a highly efficient, InlAB-independent internalization into tumor cell lines over-expressing HER1 and HER2, respectively, but not into cell lines lacking these receptors. In a xenograft murine tumor model we could also observe a significant increase in tumor colonization of this Lm strain after intravenous injection when the respective antibody was covalently crosslinked to the surface-exposed SPA.