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

Abstract

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 celltargeting bacteria Lactococcus lactis. They displayed, on their surface, binding proteins for cancerassociated 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.

Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

“…Six gene expression cassettes composed of promoter, gene of interest, and transcription terminator were selected for the assembly and were used to test the functionality of modified BglBrick system. Model proteins were either protein binders that were expressed and subsequently displayed on the surface of L. lactis [affitin against EpCAM, affibody against HER2 (Plavec et al, 2021) We successfully constructed 8 pNBBX plasmids that contained various combinations of two gene cassettes and 14 pNBBX plasmids that contained combinations of three gene cassettes, whereby both upstream and downstream cloning was used. Correct plasmid assembly was confirmed by nucleotide sequencing.…”

Dual Functionalized Lactococcus lactis Shows Tumor Antigen Targeting and Cytokine Binding in Vitro

“…Plasmid pNZDual contains two multiple cloning sites (MCS), each preceded by a nisin promoter, which enables expression of two proteins in L. lactis (Berlec et al, 2018). To construct dual expression plasmids that encode tumor antigen binder along with cytokine binder, we used pNZDual plasmids which already contained expression cassette with the tumor antigen binder gene (affi or zher) in MCS 1 and IRFP in MCS 2 [prepared previously in (Plavec et al, 2021b)], referred to as pNZ-AFFI-IRFP and pNZ-ZHER-IRFP, respectively. We replaced IRFP in the MCS 2 with the expression cassette containing the cytokine binder gene (eva or zil) via NdeI/XhoI restriction sites.…”

“…Bacterial cell adhesion assays were carried out as reported previously (Plavec et al, 2021b). Here, the medium from transfected HEK293 cells was aspirated and 500 μl 8 × 10 8 CFU/ml engineered L. lactis bacteria (diluted in pre-warmed RPMI; A 600 , 0.8) were added to each well, and incubated for 2 h at 37 °C.…”

“…The schematic of the dual gene constructs and the resulting L. lactis strains are shown in Figure 2. A FLAG-tag consensus sequence was added previously between the secretion signal and the ZHER coding sequence to facilitate detection, whereas it was excluded from the AFFI fusion protein due to negative effect on its display and functionality (Plavec et al, 2021b).…”

“…Small nonimmunoglobulin scaffold proteins, EpCAM-binding affitin (Kalichuk et al, 2018), and HER2-binding affibody (Feldwisch et al, 2010), that possess high specificity and picomolar affinity for their receptors served as the targeting ligands. L. lactis bacteria that displayed these tumor antigen ligands on their surface were recently shown to specifically bind to tumor surface antigens (Plavec et al, 2021b). Lectin-displaying L. lactis that target carbohydrate receptors on cancer cells were also developed (Plavec et al, 2021c).…”