Abstract
In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH "heterodimers." As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics.
Highlights
We sought to engineer highly efficacious agents that neutralize ricin toxin
In light of our recent success in developing VHH antibodies against botulinum neurotoxins (BoNTs) and Stx, we propose that this antitoxin technology platform may have important applications for biodefense
VHH Heterodimers Achieve Higher Affinity and Neutralizing Potency in Vitro—In the case of BoNT and Shiga toxins 1 (Stx1) and Stx2, we have demonstrated that heterodimers created by covalently linking two different toxin-neutralizing VHH monomers resulted in bi-specific antibodies with increased toxin-specific affinities and improved toxin-neutralizing activities [21, 22]
Summary
We sought to engineer highly efficacious agents that neutralize ricin toxin. Results: We identified monomeric single-chain camelid VH domains (VHHs) capable of neutralizing ricin in vitro and engineered heterodimeric VHHs that neutralized ricin in vivo. Conclusion: Stepwise engineering of VHHs resulted in highly potent ricin toxin-neutralizing antibodies. In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH “heterodimers.” As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. The two additional ricinspecific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics
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