Project Summary/Abstract Inflammatory bowel disease (IBD), including the two most common subtypes: Crohn’s disease (CD) and ulcerative colitis (UC), represents a group of intestinal disorders that cause prolonged inflammation of the digestive tract. The current therapeutic strategies, including the conventional anti-inflammatory medications and the new biologic drugs targeting the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα), have limited therapeutic efficacy and adverse drug reactions resulting from systemic administration. Colon-targeted oral delivery of anti-TNFα agents is highly desirable for the treatment of IBD, as it improves the drugs’ efficacy while reducing the systemic toxicity. Plant cell culture has emerged as a safe and cost-effective bioproduction platform for therapeutic proteins. A unique feature of the plant cells is that they could serve not only as the “bio-factory,” but also the oral delivery vehicle for recombinant biologics. Recent advances have demonstrated that plant cell walls, made primarily of cellulose microfibrils, can act as an excellent natural capsule for the oral delivery of biologic drugs. This project aims to leverage two unique posttranslational modifications – “glycosyl- phosphatidylinositol (GPI) anchor” and “plant-specific hydroxyproline (Hyp)-O-glycosylation” – to strategically design and engineer novel anti-TNFα biomolecules in plant cells to develop a new class of oral biologic drugs for the treatment of UC. The designer anti-TNFα biomolecules consist of three functional domains: a N-terminal single-chain fragment variable (scFv) of an anti-TNFα antibody, a proprietary Hyp-O-glycosylation module comprised of tandem repeats of the “Ser-Pro” motif, or (SP)n (n= 5 to 30), and a C-terminal GPI anchor. While the GPI anchor will “display” the expressed anti-TNFα biomolecules at the plant cell surface (but still encapsulated within the cell wall) to presumably create a high local concentration of the biologics, the (SP)n glycomodule will stabilize the protein from degradation during both the bioproduction and oral delivery processes. Meanwhile, the negatively charged glycans decorated on the (SP)n glycomodule will target the anti-TNFα biomolecules to the inflamed epithelium where positively charged proteins are always built up. Designer anti- TNFα biomolecules consisting of different sized (SP)n glycomodules will be investigated for their accumulation in tobacco BY-2 cells, biological activity, and stability in a simulated gastric fluid, which will determine an optimal design for the biomolecules. In order to improve the pharmacokinetic behavior of the plant cell produced oral biologic drugs, strategies will be developed to reinforce the plant cell wall matrices by filling in the cell wall pores/channels with polymeric molecules, such as polyethylene glycol (PEG), to increase their capacity for protecting the encapsulated proteins. Finally, the therapeutic effectiveness of the orally administrat...