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

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.

Keywords: antibody engineering; biodefense; immunology; mouse; neutralizing; ricin; single-domain antibody (sdAb,nanobody); therapeutic; toxin.

FIGURE 1.

Neutralization of ricin toxin by VHH heterodimers and homodimers in a mouse model. A, VHH heterodimer JJX21, and B, mAb PB10 were each administered to adult BALB/c mice (n = 5 mice per group) 2 h prior (t = −2 h), concurrent with (t = 0), or 2 h after (t = +2 h) a 10× LD₅₀ of ricin challenge. Survival was monitored for 8 days. C, VHH heterodimers (JNA8, JNA10, and JNA11), or D, VHH homodimers (JNA2, JNA3, JNA6, and JNA14) were mixed with 10× LD₅₀ of ricin at an 8:1 molar VHH:toxin ratio, and then injected intraperitoneally into BALB/c mice (n = 5 mice per group). Survival was monitored for a total of 8 days. Antibody PB10 was used as a positive control for these studies, as described under “Experimental Procedures.” Panels A and B correspond to a single experiment with shared controls, but were separated for clarity. It should be noted that VHH constructs JNA8, JNA2, JNA3, JNA6, and JNA14 significantly extend time to death, as compared with toxin-only treated mice (p < 0.05; Log-Rank Mantel-Cox test).

FIGURE 2.

Recognition of soluble ricin and ricin subunits by bispecific VHH heterodimers. Panels A, D, and G, direct ELISAs in which microtiter plates were coated with RTA or RTB, as indicated, and then probed with 2-fold serial dilutions of JNA10 (A), JNA11 (D), or JNA8 (G). Optical density (OD) at 450 nm is shown on the y axis. Panels B, C, E, F, H, and I, competition ELISA in which microtiter plates were coated with ricin and then probed with VHH heterodimers (B and C) JNA10, JNA11 (E and F), or JNA8 (H and I) in the presence of 2-fold serial dilutions of soluble ricin (middle panels), RTA or RTB (right panels). The percent of antibody bound (captured) to the plate is indicated on the y axis. The results represent a single representative experiment done in triplicate with each point indicating mean ± S.D. In some instances error bars are masked by symbol and therefore not visible.

FIGURE 3.

In vitro neutralization of ricin toxin by VHH heterodimers and homodimers. Ricin was mixed with serial dilutions of VHH heterodimers (A) JNA8, JNA10, or JNA11 or (B) homodimers JNA2, JNA3, or JNA6 and then applied to Vero cells, as described under “Experimental Procedures.” Cell viability was measured 48 h later. The results represent a representative experiment done in triplicate with each data point indicating mean ± S.D. Each agent was tested at least twice.

FIGURE 4.

Recognition of soluble ricin and ricin subunits by VHH homodimers. ELISA plates were coated with ricin and then probed with VHH homodimers JNA6 (A–C), JNA2 (D–F), or JNA3 (G–I) in the presence of 2-fold serial dilutions of soluble ricin (left panels), RTA (middle panels), or RTB (right panels). The percent of antibody bound to the plate is indicated on the y axis. The results represent a single representative experiment done in triplicate with each data point indicating mean ± S.D. In most instances error bars are masked by symbols and therefore not visible.

FIGURE 5.

Sedimentation coefficients for ricin and ricin-VHH complexes. Ricin (green), VHH constructs (black), or ricin-VHH complexes (red) were subject to AUC, as described under “Experimental Procedures.” Each panel corresponds to a single VHH construct, as indicated in the label. The ricin-VHH mixtures were incubated at room temperature for 1 h before being then subjected to AUC (20 °C). For convenience, the corrected sedimentation coefficients (s_20,w) are denoted above each sedimentation distribution in each plot. The AUC profiles are qualitative in nature and the values associated with the individual x- and y-axes are adjusted to maximize the visible distributions. VHH homodimers JNA2 and JNA6 are shown in panels A and B, pseudo-homodimer in panel C, VHH heterodimers in panels D–F, and a representative VHH monomer in panel G.

FIGURE 6.

Inhibition of ricin binding to cell surfaces by heterodimeric and homdimeric VHHs. VHH heterodimers and homodimers were mixed with FITC-labeled ricin prior to being applied to THP-1 cells in solution and then subjected to flow cytometry, as described under “Experimental Procedures.” The percent binding was normalized to ricin only treated cells (solid black bar). Lactose was used as a known inhibitor of ricin attachment (gray bar). Experiments were repeated three independent times and the bar graphs represent each treatment mean ± S.D., values are shown by each corresponding treatment. One-way analysis of variance was used to compare the ricin control to each antibody treatment (**, p < 0.01; ***, p < 0.001).