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.

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies

“…Determining VHH Specificity Using Competition ELISAsCompetition ELISAs were performed as described previously (11). In brief, Nunc Immuno MicroWell 96-well plates from ThermoFisher Scientific (Rochester, NY) were coated overnight with 0.1 g/well of ricin (15 nM) in PBS (pH 7.4).…”

“…Sedimentation Velocity Analytical Ultracentrifugation (AUC)-Ricin and VHHs were mixed at a 1:1 molar ratio and 2-fold dilutions were made, as previously described (11). Samples were mixed for at least 1 h before being subjected to AUC and all three dilution samples were run in parallel.…”

“…The five RTA-and one RTB-specific VHHs were each able to neutralize ricin toxin in vitro, albeit with varying IC 50 values. However, the monomeric VHHs were unable to neutralize ricin in vivo; when passively administered to mice, the monomeric VHHs, alone or in combination with other VHHs did not protect animals from ricin intoxication, although in some cases they extended time to death (11,12). This was true even when VHH monomers were administered at 100:1 VHH:toxin stoichiometric ratios.…”

“…Although a number of promising toxinneutralizing murine and humanized monoclonal antibodies (mAbs) are in the pipeline, the emergence of technologies surrounding the generation and expression of toxin-specific camelid heavy chain-only VH domains (VHHs) 3 has opened up new avenues for the rational design and delivery of antitoxin agents (5). VHHs with toxin-neutralizing activity have been described against botulinum neurotoxin (6), Clostridium difficile toxins TcdA and TcdB (7)(8)(9), Shiga toxins (10), ricin (11,12), and anthrax toxin (13). X-ray crystallography of toxin-VHH complexes has provided novel insights into the mechanisms of toxin neutralization (7,14) and single chain antibodies have been successfully delivered into the intracellular compartment of mammalian cells where they can effectively inactivate their targets (15).…”

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

“…Five (4 RTA-specific and 1 RTB-specific) V H Hs among the original 20 had strong or moderate in vitro toxin-neutralizing activity (TNA), while the remaining 15 had weak TNA or no detectable TNA. A collection of homo-and heterodimeric (i.e., bispecific) V H Hs were engineered with these original 20 V H Hs and shown to have enhanced avidities for ricin and, in some cases, the capacity to passively protect mice from lethal-dose ricin challenge (12)(13)(14)(15). As discussed below, the X-ray crystal structures of 10 different V H H-RTA complexes, including 7 V H Hs from the original screen (16)(17)(18), have been solved to date.…”