BackgroundBotulinum 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 FindingsWe 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.ConclusionsAn 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 of an antibody antidote for BoNT exposure.

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Immune complexes formed between monoclonal antibodies (mAbs) and toxins can neutralize toxicity in vivo by multiple mechanisms. Toxin sequestration and clearance by mAbs may be improved by enhancing their ability to bind to red blood cells (RBCs) through immune adherence. This can be achieved by converting the mAbs to heteropolymers (HPs), which are antigen-specific mAbs cross-linked to mAbs targeting the complement receptor (CR1), a protein that is expressed on the surface of RBCs in primates and mediates delivery of complement C3b-containing immune complexes to tissue macrophages. Conversion of mAbs to HPs has been shown to enhance clearance of multivalent antigens from the blood circulation, but the interaction of HPs with monovalent toxins has not been examined. Using botulinum neurotoxin (BoNT) as a model system, we studied the effect of conversion of a pair of BoNT-specific mAbs into HPs on toxin neutralization and handling in vivo. Two HPs given in combination had 166-fold greater potency than un-modified mAbs, neutralizing 5,000 LD50 BoNT, when tested in transgenic mice expressing human CR1 on RBC membranes. Improvement required adherence of BoNT to the RBC in vivo and 2 HPs, rather than an HP + mAb pair. The HP pair bound BoNT to RBCs in the circulation for 2 hours, in comparison to BoNT-neutralizing anti-serum, which induced no detectable RBC binding. HP pairs exhibited enhanced uptake by peritoneal macrophages in vitro, compared to pairs of mAbs or mAb + HP pairs. In a post-exposure therapeutic model, HPs gave complete protection from a lethal BoNT dose up to 3 hours after toxin exposure. In a pre-exposure prophylaxis model, mice given HP up to 5 days prior to BoNT administration were fully protected from a lethal BoNT dose. These studies elucidate general mechanisms for the neutralization of toxins by HP pairs and demonstrate the potential utility of HPs as BoNT therapeutics.

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.

Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both A␤ and Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both A␤ and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble A␤ in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal A␤ amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.