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.
Highlights
There are ongoing initiatives to develop countermeasures against ricin, a Select Toxin, as classified by the Centers for Disease Control and Prevention (CDC), and which has been the subject of a number of recent high profile bioterrorism incidents in the United States [1,2]
Following endocytosis, Ricin Toxin’s Binding Subunit (RTB) mediates the retrograde transport of RTA from the plasma membrane to the trans-Golgi network (TGN) and endoplasmic reticulum (ER), where RTA is liberated from RTB and retro-translocated into the cell cytoplasm [8,9]
3.1 Passive Protection Studies with VHH RTB-B7 We previously reported that three different heterodimeric
Summary
There are ongoing initiatives to develop countermeasures against ricin, a Select Toxin, as classified by the Centers for Disease Control and Prevention (CDC), and which has been the subject of a number of recent high profile bioterrorism incidents in the United States [1,2]. Ricin is a glycoprotein derived from the castor bean plant, Ricinus communis, and a member of the medically important family of A-B toxins [3]. Ricin’s enzymatic subunit (RTA) is an RNA N-glycosidase that inactivates eukaryotic ribosomes by catalyzing the hydrolysis of a universally conserved residue within the so-called sarcin/ricin loop (SRL) of 28S rRNA [4,5]. Ricin’s B subunit (RTB) is a galactose- and N-acetylgalactosamine (Gal/GalNAc)-specific lectin that has two important functions in cytotoxicity. RTB promotes ricin attachment and endocytosis of ricin into all mammalian cell types, including epithelial cells, sinusoidal endothelial cells, and macrophages [6,7]. Following endocytosis, RTB mediates the retrograde transport of RTA from the plasma membrane to the trans-Golgi network (TGN) and endoplasmic reticulum (ER), where RTA is liberated from RTB and retro-translocated into the cell cytoplasm [8,9]
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