title: Epitopes and Mechanism of Action of the Clostridium difficile Toxin A-Neutralizing Antibody Actoxumab.
abstract: The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Lorraine D
authors.initials: LD
authors.lastname: Hernandez
authors.affiliation: Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
authors.forename: Heather K
authors.initials: HK
authors.lastname: Kroh
authors.affiliation: Merck & Co., Inc., Palo Alto, CA, 94304, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Hsieh
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Yang
authors.affiliation: Merck & Co., Inc., Palo Alto, CA, 94304, USA.
authors.forename: Maribel
authors.initials: M
authors.lastname: Beaumont
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Payal R
authors.initials: PR
authors.lastname: Sheth
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: DiNunzio
authors.affiliation: Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
authors.forename: Stacey A
authors.initials: SA
authors.lastname: Rutherford
authors.affiliation: Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Ohi
authors.affiliation: Merck & Co., Inc., Palo Alto, CA, 94304, USA.
authors.forename: Grigori
authors.initials: G
authors.lastname: Ermakov
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.affiliation: Merck & Co., Inc., West Point, PA, 19446, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Secore
authors.affiliation: Merck & Co., Inc., West Point, PA, 19446, USA.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Karczewski
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Fred
authors.initials: F
authors.lastname: Racine
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Mayhood
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Fischer
authors.affiliation: Merck & Co., Inc., Boston, MA, 02115, USA.
authors.forename: Xinwei
authors.initials: X
authors.lastname: Sher
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA.
authors.forename: Pulkit
authors.initials: P
authors.lastname: Gupta
authors.affiliation: Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, 07033, USA. Electronic address: ATherien@inceptionsci.com.
authors.forename: Alex G
authors.initials: AG
authors.lastname: Therien
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D057134:Antibodies, Neutralizing / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D000080908:Broadly Neutralizing Antibodies; D004768:Enterotoxins / Q000037:antagonists & inhibitors*; D000939:Epitopes / Q000378:metabolism*; D066329:Protein Aggregates; D011485:Protein Binding
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D001427:Bacterial Toxins; D000080908:Broadly Neutralizing Antibodies; D004768:Enterotoxins; D000939:Epitopes; D066329:Protein Aggregates; C041348:tcdA protein, Clostridium difficile; C000613979:actoxumab
keywords: Clostridium difficile infection; TcdA; epitope mapping; monoclonal antibody; toxin neutralization