Preclinical validation of oral therapeutic lead proteins targeting epithelial GM1 ganglioside for ulcerative colitis therapy

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PROJECT SUMMARY Currently, there is no curative medication available for ulcerative colitis (UC), a type of inflammatory bowel disease affecting the innermost mucosal layer of the rectum and the colon. Although mucosal healing is a major treatment goal, many patients fail to achieve mucosal healing with available UC drugs such as anti-inflammatory, immuno-modulatory and anti-TNF agents. Since epithelial repair is a critical process towards mucosal healing, an agent facilitating this process will provide a novel therapeutic strategy differentiating from existing clinical care for UC. Our therapeutic lead is a variant of the oral cholera vaccine antigen cholera toxin B subunit (CTB), which was modified with a C-terminal extension including an endoplasmic reticulum retention motif (CTBSEKDEL). We have recently shown that oral administration of CTBSEKDEL, but not native CTB, facilitates colon epithelial repair and mucosal healing in a dextran sodium sulfate (DSS)-induced acute colitis mouse model. Moreover, biweekly oral administration of CTBSEKDEL significantly reduced tumorigenesis in the azoxymethane/DSS model of colitis- associated cancer. Based on these findings, we hypothesize that CTBSEKDEL provides a prototype oral biologic facilitating mucosal healing in UC. The goal of this translational R01 project is to optimize and validate the therapeutic potential of CTBSEKDEL in preclinical UC models. Since CTBSEKDEL has already shown feasibility in an acute colitis model, we will immediately proceed with further validation in chronic colitis models. In parallel, in Aim 1, we will create CTBSEKDEL variants with modifications in the C-terminal sequence (CTB(X)H/KDEL) to improve molecular stability upon spray dry for enteric-coated formulations. We will produce these proteins using a Nicotiana benthamiana plant transient overexpression system and screen them based on a series of biochemical and biophysical assays, as well as a mouse acute DSS colitis model. In Aim 2, we will validate the efficacy and safety of CTBSEKDEL and a selected CTB(X)H/KDEL in two chronic colitis models based on repeated DSS exposure in C57bl/6 mice and piroxicam-exposed IL-10 knockout mice, in comparison to an anti-TNFα antibody. Therapeutic efficacy and safety will be determined by disease activity index, histopathology, immunohistochemistry and molecular biological analysis of inflammation, crypt regeneration, epithelial barrier recovery and fibrosis. In Aim 3, we will employ a human colon explant model to further validate the mucosal healing potential of CTBSEKDEL and CTB(X)H/KDEL. Colon biopsy and colectomy tissues will be obtained from patients with different disease history and biological backgrounds. Efficacy will be evaluated based on wound healing-related gene expression and immunohistochemistry for epithelial proliferation/regeneration markers. Cell type-specific responses will be investigated in colon lamina propria mononuclear cells and colonic organoids. Collectively, the project will generate pivotal preclinical data supporting the development of a first-in-class oral biologic candidate inducing colon epithelial repair for UC treatment towards a Phase I clinical trial.