The process of intoxication proceeds by a number of steps, generally beginning with either oral or inhalational exposure. mAbs into HPs on toxin neutralization and handlingin 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 RBCin vivoand 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 macrophagesin vitro, compared to pairs of mAbs or mAb + HP pairs. In a post-exposure therapeutic model, HPs gave complete protection from MA-0204 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. Keywords:botulinum neurotoxin, monoclonal antibody, immune adherence, Type 1 complement receptor, heteropolymer, peritoneal macrophage, toxin clearance == 1. Introduction == Botulinum neurotoxins (BoNT) are a serologically diverse family of molecules produced by organisms of the genusClostridium. BoNTs are the most potent biological toxins known and have been designated as category A select bioterror agents (Arnon et al., 2001). BoNTs induce peripheral neuromuscular and autonomic paralysis by inhibiting cholinergic function. The process of intoxication proceeds by a number of steps, generally beginning with either oral or inhalational exposure. BoNT crosses the intestinal or respiratory epithelium and then transits through the blood circulation to reach its target sites, cholinergic nerve endings at neuromuscular junctions (NMJ) (Simpson, 2013). At the NMJ, IL6R BoNT is internalized by the presynaptic neuron through endocytosis. Within the neuron, the BoNT catalytic light chain domain exits the endocytic vesicle and enters the cytoplasm, where it cleaves proteins that are required for the release of acetylcholine in response to MA-0204 neuronal stimulation. Once BoNT has been internalized by a nerve ending and has cleaved its substrate, the nerve ending is no longer functional. Therefore, BoNT countermeasures need to prevent interaction of the toxin with cholinergic nerve endings. Methods that use monoclonal antibodies (mAbs) to sequester BoNT in the blood circulation and enhance clearance can contribute to BoNT neutralization by interfering with a key step in BoNT intoxication. Because BoNT exists in 7 known serotypes and multiple sub-serotypes that can differ dramatically in mAb binding and sensitivity, a comprehensive biodefense preparedness strategy for BoNT exposure may require dozens of different mAbs (Hill et al., 2007;Smith et al., 2005). The primary motivation for the present study is that mAbs capable of binding to multiple BoNT serotypes appear to be less potent at neutralization than single serotype-specific mAbs, so optimizing BoNT sequestration and clearance may be important for creating a definitive, poly-specific BoNT therapeutic (Garcia-Rodriguez et al., 2011). Antibody binding induces rapid clearance of BoNT from the bloodstream through sequestration of BoNT in the liver and spleen (Ravichandran et al., 2006). Clearance requires binding of polyclonal antiserum or at least three distinct antibodies (L. Simpson and F. Al-Saleem, unpublished observations) (Nowakowski et al., 2002;Ravichandran et al., 2006). The mechanism is extremely potent, with a capacity of neutralizing > 10,000 LD50 BoNT, and occurs within minutes of intravenous injection (Nowakowski et al., 2002;Ravichandran et al., 2006). This clearance can also be induced with polypeptide-tagged single-chain variable fragments (scFv) that form immune complexes when mixed with a mAb specific for the polypeptide tag (Sepulveda et al., 2010). The mechanism for clearance of BoNT in an immune complex likely involves capture by Fc receptor-bearing fixed tissue macrophages (Takai, 2005). Complement-mediated mechanisms may contribute to this process, as a study in humans showed that a proportion of antibody-containing immune complexes can incorporate complement C3b and adhere to red blood cells (RBCs) through complement receptor type 1 (CR1) (Davies et al., 1990). The ability of mAbs to sequester antigens in the blood circulation and deliver them to fixed tissue macrophages can be enhanced by directly binding them to RBCs through CR1 binding. Heteropolymers (HPs) are cross-linked mAb MA-0204 MA-0204 complexes in which one of the mAbs.