title: Lactic Acid Bacteria with Concomitant IL-17, IL-23 and TNFα- Binding Ability for the Treatment of Inflammatory Bowel Disease.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biotechnology, Jozef Stefan Institute, Jamova 39, SI-1000, Ljubljana. Slovenia.
authors.forename: Stasa
authors.initials: S
authors.lastname: Kosler
authors.affiliation: Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana. Slovenia.
authors.forename: Borut
authors.initials: B
authors.lastname: Strukelj
authors.affiliation: Department of Biotechnology, Jozef Stefan Institute, Jamova 39, SI-1000, Ljubljana. 0.
authors.forename: Ales
authors.initials: A
authors.lastname: Berlec
mesh_terms: D000284:Administration, Oral; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000628:therapy*; D020381:Interleukin-17 / Q000037:antagonists & inhibitors*; D053759:Interleukin-23 / Q000037:antagonists & inhibitors*; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D019936:Probiotics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
chemical_list: C508717:IL17A protein, human; D020381:Interleukin-17; D053759:Interleukin-23; D011993:Recombinant Fusion Proteins; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha
keywords: IL-17A; IL-23; Inflammatory bowel disease; TNFα; probiotics; recombinant