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

Cristina Herrera,Jacqueline M Tremblay,Charles B Shoemaker,Nicholas J Mantis

doi:10.1074/jbc.m115.658070

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.

Highlights

Bispecific heterodimeric camelid heavy chain-only antibodies (VHHs) have been shown to neutralize ricin toxin in vivo
Heterodimer VHH-based neutralizing agents (VNAs) consisting of an anti-RTB VHH (RTB-B7) linked to one of three different ricin toxin A (RTA)-specific VHHs (RTA-D10, RTA-E5, and RTAF5) were each able to neutralize ricin in vivo at VHH:toxin stoichiometric ratios as low as 4:1, thereby making them as effective as the most potent murine monoclonal antibodies (mAbs) described to date [16]
Engineering New Bispecific VHH Heterodimers Consisting of Neutralizing and Weakly Neutralizing Monomeric Constituents—We previously reported that three different VHH heterodimers (JJX12, JJX3, and JJX21), each consisting of potent toxin-neutralizing, RTA- and RTB-specific monomers, were able to protect mice from lethal dose ricin challenge when co-injected with toxin [11, 12]

Summary

Background

Bispecific heterodimeric camelid heavy chain-only antibodies (VHHs) have been shown to neutralize ricin toxin in vivo. The VHH heterodimers that were most effective at promoting ricin aggregation in solution were the most effective at blocking ricin attachment to cell surfaces 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. Heterodimer VNAs consisting of an anti-RTB VHH (RTB-B7) linked to one of three different RTA-specific VHHs (RTA-D10, RTA-E5, and RTAF5) were each able to neutralize ricin in vivo at VHH:toxin stoichiometric ratios as low as 4:1, thereby making them as effective as the most potent murine mAbs described to date [16] It was not determined whether the bivalent and/or the bispecific nature of VNAs was critical in modulating toxin neutralizing activity in the mouse model. We propose that heterodimeric VNAs may neutralize ricin in vivo through the formation of antibody-toxin complexes and thereby impair the ability of ricin to access host cell surfaces

Experimental Procedures

Results

Discussion