Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

Background: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components o

Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo

BoNT neutralization activity of the 4LCA human monoclonal antibody in the mouse protection assay.

*<p>mice were healthy at 30 days.</p

4LCA variable domain usage, CDR3 sequences, and mutation status.

<p>CDR3, complementary determining region 3. Muts, mutations in the entire IgG HC and LC variable domains.</p

Kinetic exclusion analysis (KinExA) of the solution binding properties of the 4LCA antibody and BoNT/A LC.

<p>(A) The equilibrium binding affinity was determined by titrating recombinant BoNT/A LC into solutions of fixed 4LCA concentration. At equilibrium, free 4LCA was captured by passage through a bead matrix conjugated to LC and measured by binding of a fluorescent secondary antibody. The data were fit using the manufacturer's software to a bimolecular binding model, giving an optimized KD value of 31±5×10⁻¹² M. (B) The association rate constant (k_on) was measured by mixing 4LCA (200 pM) with LC (10 pM) and measuring the concentration of free antibody concentration over time. The average k_on value for 4LCA was estimated to be 2.3±0.1×10⁶ M⁻¹ s⁻¹, which gives a calculated k_off value of 7.1×10⁻⁵ s⁻¹.</p

The effect of BoNT/A-specific antibodies on endocytosis of BoNT/A by Neuro-2a cells.

<p>Neuro-2a cells were cultured with fluorescently-labeled BoNT/A (red), with or without human antibody (green), and visualized by confocal microscopy. Panel (A) No antibody. (B) 6A antibody. (C) 4LCA antibody. In each panel, 4 images of each field are shown: BoNT/A (BT), Antibody (AB), merged (M), and phase contrast (P).</p

Effect of human antibodies on cleavage of SNAP-25 by BoNT/A in Neuro-2a cells.

<p>Neuro-2a cells were incubated with BoNT/A for 48 hours in the presence or absence of human antibody. (A) Cleavage of SNAP-25 was detected by immunoblotting of whole cell extracts. Removal of a 9 amino acid C-terminal fragment gives a band of reduced size (Cleaved) in addition to an intact SNAP-25 band (Uncleaved). Cells received either the antibodies indicated or culture medium without antibody (M). (B) The extent of SNAP-25 cleavage in the experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003023#pone-0003023-g002" target="_blank">Figure 2A</a> was quantified by scanning densitometry. Values are reported normalized to the cell sample that contained BoNT/A but no antibody.</p

Plasma BoNT/A concentrations in mice injected with biotinylated anti-BoNT antibodies with or without FP.

<p>Groups of three mice each were injected i.v. with 6 µg of inactivated BoNT/A, in the presence of the indicated combinations of biotinylated mAbs 6A and 4LCA (3 µg each) and FP (24 µg). After 90 minutes, blood was collected and plasma BoNT/A concentrations were determined by ELISA.</p