Toxin-specific antibodies for the treatment of Clostridium difficile: current status and future perspectives

Abstract

Therapeutic agents targeting bacterial virulence factors are gaining interest as non-antibiotic alternatives for the treatment of infectious diseases. Clostridium difficile is a Gram-positive pathogen that produces two primary virulence factors, enterotoxins A and B (TcdA and TcdB), which are responsible for Clostridium difficile-associated disease (CDAD) and are targets for CDAD therapy. Antibodies specific for TcdA and TcdB have been shown to effectively treat CDAD and prevent disease relapse in animal models and in humans. This review summarizes the various toxin-specific antibody formats and strategies under development, and discusses future directions for CDAD immunotherapy, including the use of engineered antibody fragments with robust biophysical properties for systemic and oral delivery.

Keywords: Clostridium difficile; Clostridium difficile-associated disease; antibody; neutralization; single-domain antibody; therapy; toxin.

Figure 1

Schematic representation of Clostridium difficile toxin A and B. For illustration purposes, only one toxin is shown. Toxin A (TcdA, 308 kDa) and toxin B (TcdB, 269 kDa) are each composed of four domains, which perform distinct functions. The schematic illustrates each domain, their function, and site of action. GT = glucosyltransferase domain, CP = cysteine protease domain, MI = hydrophobic membrane insertion domain, RBD = cell-receptor binding domain.

Figure 2

Schematic illustration of Clostridium difficile toxin mechanism of action. TcdA or TcdB first binds the surface of epithelial cells via the RBD region of the toxin, promoting receptor-mediated endocytosis. Acidification of the endosome-encapsulated toxin promotes a conformational change in which the N-terminal region of the toxin is extended through the endosomal membrane into the cytoplasm. Cellular inositol hexakisphosphate (InsP6) promotes cleavage at the start of the CP domain, releasing the GT domain into the cytoplasm. The GT domain transfers a glucose moiety from UDP-glucose to a threonine (T) residue on Rho-GTPase, trapping the signaling enzyme in an inactive conformation. Targeting the RBD domain with antibodies and antibody fragments may block toxin binding to cell-surface receptors or prevent internalization of the toxin.

Figure 3

Various antibody formats for anti-toxin therapy. Traditional antibody formats (i.e., IgY, IgG, IgA) targeting C. difficile toxins have been produced primarily from immunized animals. Smaller recombinant antibody binding fragments (i.e., Fab, scFv, V_L, V_H, V_HH) produced from in vitro selection procedures may be useful agents to explore for CDAD immunotherapy. Of these recombinant fragments, single-domain antibodies (i.e., V_HH) from Camelidae heavy-chain IgGs possess inherent thermal and protease stability and have been shown to bind cryptic epitopes or pockets on proteins that cannot be accessed by traditional antibodies. As such, these single-domain antibodies may be potent toxin neutralizers and promising therapeutic agents for CDAD immunotherapy. Black bars represent disulfide bonds, grey bars represent hinge regions, and the red bar represents a synthetic linker. Some Igs have more than two inter-heavy chain disulfide linkages.