title: Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.
abstract: Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors.
authors.affiliation: From the Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York 12208, the Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York 12201, and.
authors.forename: Cristina
authors.initials: C
authors.lastname: Herrera
authors.affiliation: the Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachuetts 01536.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Tremblay
authors.affiliation: the Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachuetts 01536.
authors.forename: Charles B
authors.initials: CB
authors.lastname: Shoemaker
authors.affiliation: From the Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York 12208, the Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York 12201, and nicholas.mantis@health.ny.gov.
authors.forename: Nicholas J
authors.identifier: http://orcid.org/0000-0002-5083-8640
authors.initials: NJ
authors.lastname: Mantis
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000276:immunology; D002161:Camelids, New World / Q000276:immunology; D002522:Chlorocebus aethiops; D005260:Female; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000276:immunology; D020128:Inhibitory Concentration 50; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering; D055503:Protein Multimerization; D012276:Ricin / Q000037:antagonists & inhibitors* / Q000276:immunology; D014461:Ultracentrifugation; D014709:Vero Cells
chemical_list: D057134:Antibodies, Neutralizing; D007143:Immunoglobulin Heavy Chains; D012276:Ricin
keywords: antibody engineering; biodefense; immunology; mouse; neutralizing; ricin; single-domain antibody (sdAb,nanobody); therapeutic; toxin