title: Mechanisms of enhanced neutralization of botulinum neurotoxin by monoclonal antibodies conjugated to antibodies specific for the erythrocyte complement receptor.
abstract: 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 5000 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 2h, 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 3h 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.
authors.affiliation: Lankenau Institute for Medical Research, Wynnewood, PA 19096, USA.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Sharma
authors.forename: Huiwu
authors.initials: H
authors.lastname: Zhao
authors.forename: Fetweh H
authors.initials: FH
authors.lastname: Al-Saleem
authors.forename: Ahmed Syed
authors.initials: AS
authors.lastname: Ubaid
authors.forename: Rama Devudu
authors.initials: RD
authors.lastname: Puligedda
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Segan
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Lindorfer
authors.forename: Rodney
authors.initials: R
authors.lastname: Bermudez
authors.forename: Md
authors.initials: M
authors.lastname: Elias
authors.forename: Sharad P
authors.initials: SP
authors.lastname: Adekar
authors.forename: Lance L
authors.initials: LL
authors.lastname: Simpson
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Taylor
authors.forename: Scott K
authors.initials: SK
authors.lastname: Dessain
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology; D001905:Botulinum Toxins / Q000276:immunology*; D001906:Botulism / Q000276:immunology / Q000517:prevention & control*; D004912:Erythrocytes / Q000276:immunology*; D006801:Humans; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D011951:Receptors, Complement / Q000276:immunology*; D017463:Receptors, Complement 3b / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000936:Antigen-Antibody Complex; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D001905:Botulinum Toxins
keywords: BoNT; BoNT/A; BoNT/A recombinant 50kD C-terminal domain; Botulinum neurotoxin; CR1; FP; Fab′; HC50A; HP; HRP; Heteropolymer; Immune adherence; Monoclonal antibody; NMJ; OPD; PBS; Peritoneal macrophage; RBCs; RI-BoNT; Toxin clearance; Type 1 complement receptor; a fusion protein consisting of a streptavidin molecule and an scFv specific for glycophorin; botulinum neurotoxin; complement receptor; hCR1; heteropolymer; horseradish peroxidase; human complement receptor; i.p.; i.v.; intra-peritoneal; intravenous; mAb; mAb antigen binding domain; monoclonal antibody; neuromuscular junction; o-phenylenediamine dihydrochloride; phosphate buffered saline; recombinant inactive BoNT; red blood cells; scFv; serotype A botulinum neurotoxin; single-chain variable fragment