Abstract Text: Gut chronic inflammation is the culprit of many serious diseases and disorders, such as metabolic disorders, cancers, and neurological diseases. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNF-α) and Interleukin 17a (IL-17A) are the key immune mediators that contribute to the pathogenesis of gut inflammation. The goal of the project was to genetically engineer an over-the-counter probiotic yeast, Saccharomyces boulardii, for the constitutive secretion of a bi-specific antibody and to neutralize both of the inflammatory cytokines into a safe and convenient oral medicine for inhibiting chronic gut inflammation. We first designed two formats of the bi-specific antibody by fusing two anti-cytokine single-domain antibodies, or VHHs, with human IgG1 Fc (VHH1-Fc-VHH2 and VHH1-VHH2-Fc). Next, we tested two strong constitutive promoters for their ability to efficiently drive the expression of the bi-specific antibody in S. boulardii. Finally, we evaluated two non-antibiotic selection markers for the selection of chromosomal insertion of the bi-specific antibody genes. We found that the two promoters each drove a slightly different bi-specific antibody expression. The bioactivities between the two formats of bi-specific antibodies were similar, but the format of VHH1-VHH2-Fc yielded a higher expression in the probiotic yeast. The selection markers did not affect the overall screening nor the expression of the antibody. Based on these results, we selected a final engineered yeast strain, which was designated as FZ008, as our lead drug candidate. In the future, we will perform preclinical evaluations on FZ008 against gut chronic inflammation in transgenic mice, and eventually human trials against neurodegenerative diseases.
