Characterization of a Novel High-Affinity Monoclonal Immunoglobulin G Antibody against the Ricin B Subunit

Abstract

There is an urgent need for the development of a passive immunotherapy against the category B select agent ricin, a lethal ribosome-inactivating toxin composed of an enzymatic A subunit (RTA) and a single binding B subunit (RTB). To date, only one monoclonal antibody (MAb), a mouse immunoglobulin G (IgG1) against RTA called R70, has been deemed sufficiently potent in animal models to warrant further testing in humans. In this study, we have identified and characterized MAb 24B11, a murine IgG1 directed against RTB. In a Vero cell cytotoxicity assay, 24B11 was approximately two times more effective at neutralizing ricin than was R70. The equilibrium dissociation constants of 24B11 (K D ‫؍‬ 4.2 ؋ 10 ؊9 M) and R70 (K D ‫؍‬ 3.2 ؋ 10 ؊9 M) were virtually identical, suggesting that the difference in neutralization activity between the two MAbs was not due to differing affinities for the toxin. 24B11 blocked ricin attachment to galactoside receptors on primary mouse splenocytes and on the apical surfaces of human mucosal epithelial cell monolayers. Surprisingly, R70 also effectively interfered with ricin attachment to receptors on cell surfaces. Using a phage-displayed peptide library, we determined that 24B11 binds an epitope on RTB adjacent to, but not within, one of the two galactose binding domains. Finally, we demonstrate that R70 and 24B11, when combined, function synergistically to neutralize ricin in vitro, raising the possibility that these two MAbs could serve as a novel immunotherapeutic in vivo.

A Neutralizing Antibody to the A Chain of Abrin Inhibits Abrin Toxicity both In Vitro and In Vivo

“…Several neutralizing antibodies for biological toxins have been described earlier (5,20,24,39). Antibodies to the galactose binding domain of the B chain have been shown to inhibit ricin binding to cells, thereby inhibiting toxin activity (19,20). Antibodies to the A chain of ricin also protect cells effectively from ricin toxicity (18).…”

“…Antibody-based therapies, on the other hand, for the treatment of poisoning by various toxins are more promising. Several neutralizing antibodies for biological toxins have been described earlier (5,20,24,39). Antibodies to the galactose binding domain of the B chain have been shown to inhibit ricin binding to cells, thereby inhibiting toxin activity (19,20).…”

Functional Analysis of Neutralizing Antibodies against Clostridium perfringens Epsilon-Toxin

“…In the case of epsilon-toxin-mediated enterotoxemia, administration of toxin-neutralizing antiserum is used prophylactically to treat unvaccinated animals in the event of an outbreak of enterotoxemia (2). Epitopes recognized by antibodies that neutralize the activities of several select agent toxins have been mapped and provide valuable information that may be used in the development of novel antitoxin therapies (3,8,16,24,42,43,57,58).…”

Evidence for Widespread Epithelial Damage and Coincident Production of Monocyte Chemotactic Protein 1 in a Murine Model of Intestinal Ricin Intoxication

“…ELISA plates were developed with a one-component TMB colorimetric substrate (Kirkegaard and Perry, Gaithersburg, MD) and were read using a SpectraMax 250 microtiter plate reader equipped with Softmax software (Molecular Devices, Union City, CA). Mouse monoclonal IgGs and IgAs specific for RTA or RTB were used as controls for these assays (29,30).…”

“…In contrast, full protection was achieved when mice were immunized mucosally to stimulate SIgA antibodies in bronchoalveolar lavage fluids (13,14,20,44). We have recently produced a collection of monoclonal IgA and IgG antibodies (29,30) that when coupled with the mouse model described herein will enable us to sort out the relative contributions of the different antibody specificities and isotypes that mediate immunity to ricin in mucosal compartments. Identifying the roles of secretory IgA and IgG in mediating mucosal immunity to ricin has important implications for the delivery of a ricin vaccine to humans (31,39).…”