Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein

PLOS ONE, Mar 2011

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.

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Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein

et al. (2011) Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein. PLoS ONE 6(3): e17491. doi:10.1371/journal.pone.0017491 Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein Sharad P. Adekar 0 Andrew T. Segan 0 Cindy Chen 0 Rodney Bermudez 0 M. D. Elias 0 Bernard H. Selling 0 B. P. Kapadnis 0 Lance L. Simpson 0 Paul M. Simon 0 Scott K. Dessain 0 Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, France 0 1 Lankenau Institute for Medical Research, Wynnewood, Pennsylvania, United States of America , 2 Immunome , Inc., Wynnewood, Pennsylvania, United States of America, 3 Division of Infectious Diseases and Environmental Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America , 4 Impact Biologicals , Inc. Wallingford, Pennsylvania, United States of America, 5 Department of Microbiology, University of Pune , Pune , India , 6 Augmenta Biologicals , LLC , Wynnewood, Pennsylvania , United States of America 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 postexposure 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. - Funding: This work was supported by a grant from the National Institutes of Health, NIAID R01 AI065967 to S.K.D. and institutional support from the Lankenau Institute for Medical Research. Augmenta, LLC, provided support for PMS and contracted Impact Biologicals, Inc. for production of the FP. PMS, as a corresponding author, participated in study design, data collection and analysis, decision to publish, and preparation of the manuscript. Competing Interests: The authors have read the journals policy and have the following conflicts: SPA and RB are part-time employees of Immunome, Inc., a company that has optioned rights to the antibodies described in this report. BHS is the president of Impact Biologicals, Inc. PMS is the inventor of the FP and the President of Augmenta, LLC. SKD is a founder and Chief Scientific Officer of Immunome, Inc. and has an equity interest in the company. SKD does not receive research support or consulting fees from Immunome, Inc., Augmenta Biologicals, LLC, or Impact Biologicals, Inc. A provisional US Patent application has been filed on the experiments described, "Toxin Clearance", Inventors SPA, SKD, PMS, and has been assigned to LIMR and Augmenta, LLC. These relationships do not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. Botulinum neurotoxin is one of the most potent lethal substances known. It is produced by organisms of the genus Clostridium and produces peripheral neuromuscular and autonomic paralysis through inactivation of cholinergic signaling at the neuromuscular synapse. Intoxication with BoNT proceeds by a series of steps, in which BoNT first enters the body, transits across an epithelium, travels through the bloodstream, and interacts with the surface of cholinergic neurons [1,2,3]. Once bound to the neuromuscular junction, BoNT is internalized via binding to secretory vesicle proteins and transported into a vesicular compartment. The catalytic domain of BoNT, the light chain (LC), acquires proteolytic activity as it is transported across the vesicle membrane into the neuron cytosol [4,5]. Through cleavage of tethering proteins, the BoNT LC prevents the neuron from releasing acetylcholine in response to neural stimulation. Passive immune therapies for BoNT intoxication have b (...truncated)


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Sharad P. Adekar, Andrew T. Segan, Cindy Chen, Rodney Bermudez, M. D. Elias, Bernard H. Selling, B. P. Kapadnis, Lance L. Simpson, Paul M. Simon, Scott K. Dessain. Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein, PLOS ONE, 2011, Volume 6, Issue 3, DOI: 10.1371/journal.pone.0017491