Neutralization of Clostridium difficile Toxin A with Single-domain Antibodies Targeting the Cell Receptor Binding Domain

• Published in: The Journal of biological chemistry 2011-03, Vol.286 (11), p.8961-8976
• Main Authors: Hussack, Greg, Arbabi-Ghahroudi, Mehdi, van Faassen, Henk, Songer, J. Glenn, Ng, Kenneth K.-S., MacKenzie, Roger, Tanha, Jamshid
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antibodies, Bacterial - genetics; Antibodies, Bacterial - immunology; Antibodies, Neutralizing - genetics; Antibodies, Neutralizing - immunology; Bacterial Toxins - genetics; Bacterial Toxins - immunology; Camelids, New World; Clostridioides difficile - genetics; Clostridioides difficile - immunology; Clostridium difficile; Endotoxin; Enterocolitis, Pseudomembranous - immunology; Enterocolitis, Pseudomembranous - therapy; Enterotoxins - genetics; Enterotoxins - immunology; Epitope Mapping; Epitopes - genetics; Epitopes - immunology; Humans; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Immunology; Male; Protein Structure and Folding; Protein Structure, Tertiary; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; Single-domain Antibody; Toxin A; Toxins; VHH
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.198754

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (VHHs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when VHHs were administered in paired or triplet combinations at the same overall VHH concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific VHHs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these VHHs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract.