title: Differential neutralizing activities of a single domain camelid antibody (VHH) specific for ricin toxin's binding subunit (RTB).
abstract: Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody") specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.
authors.affiliation: Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America; Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York, United States of America.
authors.forename: Cristina
authors.initials: C
authors.lastname: Herrera
authors.affiliation: Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vance
authors.affiliation: Scientific Cores, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Leslie E
authors.initials: LE
authors.lastname: Eisele
authors.affiliation: Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, United States of America.
authors.forename: Charles B
authors.initials: CB
authors.lastname: Shoemaker
authors.affiliation: Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, United States of America; Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York, United States of America.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Mantis
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D054788:Ribosome Inactivating Proteins / Q000276:immunology; D012276:Ricin / Q000276:immunology*; D014709:Vero Cells
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D007143:Immunoglobulin Heavy Chains; D012276:Ricin; D054788:Ribosome Inactivating Proteins