Background: Neutralization of proinflammatory cytokines is an established strategy in the
treatment of inflammatory bowel disease (IBD). Systemic anti-TNFα 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 TNFα 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-TNFα-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 TNFα 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.