Lactic Acid Bacteria with Concomitant IL-17, IL-23 and TNFα- Binding Ability for the Treatment of Inflammatory Bowel Disease

Background: Neutralization of proinflammatory cytokines is an established strategy in the treatment of inflammatory bowel disease (IBD). Systemic anti-TNF alpha antibodies have been used in the clinics for several years, while anti-IL-17/IL-23 antibodies have been less successful so far. We report the development of safe lactic acid bacterium Lb. salivarius with the ability to simultaneously bind IL-17A, IL-23 and TNF alpha that could be administered orally for the treatment of IBD. Method: Three different cytokine-binding non-Ig scaffolds (anti-IL-17A fynomer, anti-IL-23-binding adnectin and anti-TNFa-binding affibody) were cloned and expressed in L. lactis in fusion with lysine motif (LysM)-containing surface anchor. Fusion proteins were used for coating of Lb. salivarius. Cytokine-binding ability of bacterial cells was assayed with ELISA. Gastric stability was tested by incubation in simulated gastric juice. Results: Surface display and functionality of cytokine binding proteins in L. lactis was confirmed by the ability to remove the individual cytokines from the solution. Binding was efficient with different concentrations of cells, different cytokine concentrations and after the incubation in simulated gastric juice. The fusion protein-containing growth media of L. lactis were used for coating of Lb. salivarius in a fast and straightforward manner. Cytokine-binder-coated Lb. salivarius was able to retain individual binders or mixtures of all three binders on its surface, and also provided limited protection from proteolytic and low pH degradation. Cytokine binding capacity of mixture-coated Lb. salivarius was similar to that achieved by coating with individual binders. Conclusion: Simultaneous binding of IL-17A, IL-23 and TNFa with engineered bacteria was achieved for the first time. Oral administration of such bacteria could exert synergistic effect and thus represents an alternative strategy in the treatment of IBD.