Colorectal cancer (CRC) is one of the most common types of cancer (the third most common cancer in men and the second in women), with a high death rate and poor prognosis. Current treatment is expensive and demanding, and can result in severe side effects. Therefore, development of new treatment approaches is of great importance. The role of microbiota in CRC development has already been confirmed through maintaining intact barrier of intestinal mucosa. In contrast to pathogens, which cause inflammation, dysbiosis and concomitant CRC development, commensal bacteria prevent intestinal damage. Lactic acid bacteria (LAB) are also a part of beneficial commensal bacteria, and are recognized as safe due to their long history of use. They are also interesting as delivery vectors for therapeutic molecules. Several different systems are available for protein surface display on model LAB Lactococcus lactis. The fusion protein is attached to the bacterial surface through an anchoring domain. Most commonly applied domain is the C-terminal region of the AcmA protein (cAcmA), which anchors to the peptidoglycan through non-covalent bond. In the first part of the doctoral thesis, we screened 15 anchoring domains, which can provide non-covalent or covalent binding to the bacterial cell wall, and compared them to cAcmA. cAM12 anchor of lactococcal phage origin was determined to be equivalent to cAcmA, and to attach to a different cell surface moiety than cAcmA. Thus, it could be used as its alternative. In the second part of the doctoral thesis, we developed LAB L. lactis with surface-displayed tumor antigen-targeting proteins. Cancer cells over-express tumor antigens on their surface, which can be used to achieve targeted therapy. Colorectal cancer is characterized by the expression of various tumor antigens (1). We focused on EpCAM, HER2 and Gb3. EpCAM and HER2 have been recently exploited as diagnostic markers (2), as well as in preparation of CRC-targeted therapy (3), while expression of Gb3, on the other hand, strongly correlates with CRC progression, which makes it a relevant target (4). We designed two non-immunoglobulin targeting proteins against EpCAM and HER2, and a lectin against Gb3, the B subunit of Shiga toxin. We used pNZDual plasmid, which allows the expression of two proteins simultaneously; apart from antigen-targeting protein also a reporter infrared fluorescent protein for detection, and confirmed the simultaneous expression of both. Different types of tumor cell lines were used to evaluate adhesion of targeting L. lactis bacteria; HeLa, HEK293, in which transfection plasmid was used to achieve overexpression of tumor antigens, and the colorectal cell lines HT-29 and Caco-2. The highest L. lactis adhesion was seen for the HEK293 cells, where adhesion to their tumor antigens amounted to 39% and 67% of EpCAM-targeting and HER2-targeting bacteria, respectively. Adhesion to colorectal cells was slightly lower, but significant in comparison to control. Specific and targeted binding of developed L. lactis to selected tumor cell lines was further demonstrated by flow cytometry and a real-time system for bacteria-to-cell adhesion monitoring. Afterward, a therapeutic function was added to L. lactis. We focused on cytokine signaling, which plays a key role in promoting inflammation and thus contributes to the progression of CRC. We presented on the surface of L. lactis REX proteins, which act as inhibitors of the IL-23 receptor and consequently stop the inflammatory pathway.
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