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Lactococcus Lactis Simultaneously Displaying Protein Binders of Tumor­ Associated Antigens and Proinflammatory Cytokines for Targeted Therapy of Colorectal Cancer

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Lactococcus Lactis Simultaneously Displaying Protein Binders of Tumor­ Associated Antigens and Proinflammatory Cytokines for Targeted Therapy of Colorectal Cancer Lactococcus lactis simultaneously displaying protein binders of tumor­ associated antigens and proinflammatory cytokines for targeted therapy of colorectal cancer P­05.2­14 A. ZahirovićI, T.V. PlavecI, A. BerlecI IDepartment of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, Slovenia, Ljubljana, Slovenia Proinflammatory cytokines interleukin (IL)­6, tumor necrosis factor (TNF), and IL­8 promote the development of colorectal cancer (CRC). The delivery of cytokine­ binding proteins into the gut using tumor­targeted safe probiotic lactic acid bacteria as a vector might represent an alternative therapeutic approach in inflammation­ associated CRC. We engineered six Lactococcus lactis strains to simultaneously display a combination of small protein binder of tumor­associated antigen (HER2­ binding affibody or EpCam­binding affitin) and a binder of proinflammatory cytokine (IL­6­binding affibody, TNF­α­binding affibody or IL­8­binding evasin) on their surface. Surface display of two proteins was achieved by cloning the genes into a lactococcal plasmid for dual protein expression (constructed by doubling nisin promoter). Protein binders were fused to Usp secretion signal and AcmA anchor to enable their attachment onto the surface. The expression of HER/EpCam­binding ligands was confirmed by Western blot or flow cytometry. The functionality of cytokine­binding moieties on the bacterial surface was demonstrated by testing their ability to bind cytokines using ELISA. Engineered bacteria sequestered 80­100% of the corresponding cytokine spiked into the solution, whereby L. lactis displaying IL­6­binding affibody proved the most efficient. Furthermore, engineered bacteria were co­cultured with immunostimulated cancer cells and showed that it is able to bind cytokines released in the culture medium. Strains displaying IL­8­binding evasin removed 60­65% of IL­8 secreted by Caco­2 and 58­62% of IL­8 secreted by HT29 colorectal cancer cells. Strain displaying IL­6­binding affibody removed 92­94% of IL­6 secreted by human monocytic THP­1 cells (undifferentiated and differentiated) and up to 96% of IL­6 secreted by myeloid leukaemia U­937 cells. Adhesion of engineered bacteria to Caco­2 cells is currently being tested to assess their tumor­targeting capacity..
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