Characterization and utility of novel monoclonal antibodies to cholera toxin B subunit

Abstract

Cholera toxin B subunit (CTB) is a potent immunomodulator exploitable in mucosal vaccine and immunotherapeutic development. To aid in the characterization of pleiotropic biological functions of CTB and its variants, we generated a panel of anti-CTB monoclonal antibodies (mAbs). By ELISA and surface plasmon resonance, two mAbs, 7A12B3 and 9F9C7, were analyzed for their binding affinities to cholera holotoxin (CTX), CTB, and EPICERTIN: a recombinant CTB variant possessing mucosal healing activity. Both 7A12B3 and 9F9C7 bound efficiently to CTX, CTB, and EPICERTIN with equilibrium dissociation constants at low to sub-nanomolar concentrations but bound weakly, if at all, to Escherichia coli heat-labile enterotoxin B subunit. In a cyclic adenosine monophosphate (cAMP) assay using Caco2 human colon epithelial cells, the 7A12B3 mAb was found to be a potent inhibitor of CTX, whereas 9F9C7 had relatively weak inhibitory activity. Meanwhile, the 9F9C7 mAb effectively detected CTB and EPICERTIN bound to the surface of Caco2 cells and mouse spleen leukocytes by flow cytometry. Using 9F9C7 in immunohistochemistry, we confirmed the preferential localization of EPICERTIN in colon crypts following oral administration of the protein in mice. Collectively, these mAbs provide valuable tools to investigate the biological functions and preclinical development of CTB variants.

The Unfolded Protein Response and Its Implications for Novel Therapeutic Strategies in Inflammatory Bowel Disease

“…In mice, EPICERTIN remained detectable in the colon epithelium 24 h after oral or intrarectal administration, particularly in the base region of colon crypts. This suggests a role for colonic crypt base stem cells in EPICERTIN-induced epithelial regeneration [126]. Mechanistically, the IRE1/XBP1s pathway of the UPR, which is known to maintain ER homeostasis by regulating cell survival/apoptosis balance [33,127], appears to mediate the wound healing activity of EPICERTIN, as the IRE1 inhibitor 4µ8C or siRNA knockdown of Xbp1 abolished EPICERTIN's wound healing effect in Caco-2 cells [116].…”