Antibody coated Lm strains not only showed specific binding to tumor cell

lines but also a DZNeP cost highly efficient internalization into tumor cell lines. This internalization was clearly independent of the known InlA and/or InlB-mediated invasion machinery of Lm, as these two major invasion factors [reviewed in 18] were deleted in the antibody-coated Lm strains. Experiments showing internalization of Trastuzumab-coated beads into HER2 expressing cells indicate that the internalization may be completely independent of listerial virulence factors. The bacteria may be taken up by the host cell passively, as a consequence of receptor recycling. The cellular recycling rate of the EGF-family receptors has been shown to increase upon ligand interaction and antibody-mediated dimerization [29]. After Trastuzumab- mediated internalization Lm was able to escape into the cytosol, replicate and spread to adjacent cells as demonstrated AZD5582 manufacturer by immunofluorescence. The efficiency of these intracellular steps was comparable to that of the corresponding ΔaroA attenuated wild-type strain. Transfer of antibody-mediated targeting into xenograft mouse tumors was initially unsuccessful. Subsequent in vitro experiments revealed that the incubation of the antibody BVD-523 concentration coated bacteria with murine serum completely abrogated the specific internalization,

but this effect was largely prevented by crosslinking of the antibody to SPA on the surface of live bacteria. Crosslinking enabled also the targeting of the antibody-coated bacteria to a 4T1-HER2 xenograft mouse tumor. The number of Trastuzumab-coated bacteria in the tumor tissue increased 8 to 10-fold when compared to uncoated bacteria. Although less than 5% of these bacteria

were intracellular, the bacterial count was significantly increased relative to bacteria not coated mafosfamide with Trastuzumab. This 3-fold increase in the number of intracellular bacteria was antibody specific, since bacteria coated with a second antibody (Cetuximab), that recognizes the related receptor EGFR, did not show a significant increase compared to uncoated bacteria. The bacterial counts in liver and spleen were 2-fold increased with the Trastuzumab-coated Lm compared to the uncoated bacteria, while the Cetuximab-coated bacteria colonized liver and spleen with a similar efficiency as the uncoated ones. The humanized Trastuzumab contains a larger portion of non-mouse peptide sequences than the human/mouse chimeric Cetuximab. Thus a stronger immune reaction against Trastuzumab might lead to an enhanced uptake of bacteria coated with Trastuzumab by phagocytic cells in liver and spleen. Recently Bereta and coworkers [23] described an alternative approach of antibody-mediated targeting of bacteria whereby a single chain antibody (scFv) was expressed by Salmonella VNP20009.