Engineering designer biologics in plant cells for oral treatment of inflammatory bowel disease (IBD).

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 treatment strategies, including the conventional anti-inflammatory medications and the new biological drugs targeting the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα), have limited therapeutic efficacy and adverse drug reactions resulted from systemic administration. Colon-targeted oral delivery of anti-TNFα agents is highly desirable for the treatment of IBD, as it improves drugs' efficacy while reducing 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 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 "displays" the expressed anti-TNFα biomolecules at the plant cell surface to presumably create a high local concentration of the biologics, the (SP)n glycomodule stabilizes the protein from degradation during both the bioproduction and oral delivery processes. Designer anti-TNFα biomolecules consisting of different sizes of the (SP)n glycomodule are investigated for their accumulations in tobacco BY-2 cells, biological activity, and stability in a simulated gastric fluid, which determines the (SP)20 module as an optimal design for the biomolecules. The therapeutic effectiveness of the orally administrated designer anti-TNFα biologic (optimal design) in mitigating the UC symptom is assessed in a dextran sulfate sodium (DSS)-induced colitis mouse model. The immune-modulatory effects of the anti-TNFα biologics are determined by histopathological analysis and assay of the inflammatory markers. The research may develops a new platform to produce effective oral biologic drugs for the treatment of UC and other inflammatory diseases of colon.