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Summary  Development of targeted treatment for colorectal cancer is crucial to avoid side effects. To harness the possibilities offered by microbiome engineering, we prepared safe multifunctional cancer cell‐targeting bacteria  Lactococcus lactis . They displayed, on their surface, binding proteins for cancer‐associated transmembrane receptors epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) and co‐expressed an infrared fluorescent protein for imaging. Binding of engineered  L. lactis  to tumour antigens EpCAM and HER2 was confirmed and characterised  in vitro  using soluble receptors. The proof‐of‐principle of targeting was demonstrated on human cell lines HEK293, HT‐29 and Caco‐2 with fluorescent microscopy and flow cytometry. The highest  L. lactis  adhesion was seen for the HEK293 cells with the overexpressed tumour antigens, where colocalisation with their tumour antigens was seen for 39% and 67% of EpCAM‐targeting and HER2‐targeting bacteria, respectively. On the other hand, no binding was observed to HEK293 cells without tumour antigens, confirming the selectivity of the engineered  L. lactis . Apart from cell targeting in static conditions, targeting ability of engineered  L. lactis  was also shown in conditions of constant flow of bacterial suspension over the HEK293 cells. Successful targeting by engineered  L. lactis  support the future use of these bacteria in biopharmaceutical delivery for the treatment of colorectal cancer.

Targeting of fluorescent Lactococcus lactis to colorectal cancer cells through surface display of tumour‐antigen binding proteins