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 administrated designer
anti-TNFα biologic (optimal design) in mitigating UC symptoms will be assessed in a dextran sulfate sodium
(DSS)-induced colitis mouse model. The immune-modulatory effects of the anti-TNFα biologics will be
determined by a histopathological analysis and assay of the inflammatory markers. The proposed research will
potentially develop a new platform to produce effective oral biologic drugs for the treatment of UC and other
inflammatory diseases in the colon.
