Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins

Adekar, Sharad P.; Jones, R. Mark; Elias, M.D.; Al-Saleem, Fetweh H.; Root, Michael J.; Simpson, Lance L.; Dessain, Scott

doi:10.1016/j.jim.2008.01.015

Cited by 30 publications

(44 citation statements)

References 45 publications

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“…While polyclonal antitoxin products are available to treat human botulism, it is widely accepted that improved antitoxin agents are badly needed (27). One approach showing promise is to replace polyclonal antisera with monoclonal antibodies (MAbs) (1,2,4,9,17,20). This approach has been applied successfully in mice, using anti-BoNT MAbs (17).…”

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confidence: 99%

Efficient Serum Clearance of Botulinum Neurotoxin Achieved Using a Pool of Small Antitoxin Binding Agents

et al. 2010

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Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD 50 ) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD 50 of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.

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Efficient Serum Clearance of Botulinum Neurotoxin Achieved Using a Pool of Small Antitoxin Binding Agents

et al. 2010

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“…Polyclonal human HCs were generated from purified IgGs by mild reduction and alkylation and purified under acidic conditions using size exclusion chromatography (27). Monoclonal human HCs were generated from mAbs, 13A and 30B (28,29), by mild reduction and alkylation and purified using affinity chromatography with a column consisting of rabbit anti-human free and LC IgGs (Dako, Carpinteria, CA) conjugated to a N-hydroxysuccinimide-activated Sepharose 4 fast flow agarose matrix (GE Healthcare). The percentage of yield of each soluble HC preparation was determined by ultracentrifugation (50,000 ϫ g for 2 h at 4°C) to be ϳ50 -90% for the monoclonal HCs F1, 30B, and 13A and ϳ15-25% for the polyclonal HCs.…”

Section: Methodsmentioning

confidence: 99%

“…Splenic MCs were stored frozen in 90% heat-inactivated fetal calf serum (Invitrogen) and 10% Me 2 SO (Sigma-Aldrich) under liquid nitrogen. Prior to cell fusion, CD27-positive splenic MCs were isolated with anti-CD27 magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions, and splenic MCs were cultured for 8 days on a monolayer of tCD40L cells (courtesy of Gordon Freeman, Dana Farber/Partners Cancer Care, Boston, MA) (29).…”

Section: Methodsmentioning

confidence: 99%

“…Cultured splenic MCs (2.5 ϫ 10 6 ) were fused to the B5-6T heteromyeloma cell line, which ectopically expresses the hTERT and mIL-6 genes, at a 1:1 ratio using the stirring method with 50% polyethylene glycol (Sigma-Aldrich), plated in 20 wells of a 48-well plate (Corning), and selected in HAT medium (Sigma-Aldrich) (29). Hybridoma supernatants that were positive in EuLISA against plate-immobilized Jto fibrils were selected for subcloning by limiting dilution.…”

Section: Methodsmentioning

confidence: 99%

“…Amyloid Fibril and Oligomer Cross-reactivity Is a General Property of HCs-To establish whether amyloid fibril and oligomer cross-reactivity is a general and novel property of free human Ig ␥ HCs, we isolated HCs from two human mAbs, 13A and 30B, which are specific for botulinum neurotoxins (28,29) and do not react with amyloid fibrils. Fig.…”

Section: Table 1 Monoclonal Human Igg and Hc Binding To Amyloid Fibrimentioning

confidence: 99%

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Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

Adekar

Klyubin

Macy

et al. 2010

Journal of Biological Chemistry

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We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of A␤, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig ␥ heavy chains. A ␥ 1 heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloidladen tissue sections. F1 did not bind to native A␤ monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig ␥ heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble A␤ oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig ␥ heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules. Alzheimer disease (AD)3 is approaching global epidemic proportions and is the most common of over 25 incurable protein misfolding diseases that are termed the amyloidoses (1, 2). A hallmark of the disease is deposition of amyloid fibril-containing neuritic plaques that are formed by abnormal processing of ␤-amyloid protein (A␤), a proteolyzed transmembrane 39 -43 fragment of amyloid precursor protein (3). Diverse experimental studies support a central pathogenic role for aggregated A␤, which in the brain initiates a cascade of events that ultimately lead to neuronal dysfunction and death (4 -6). The most neurotoxic A␤ species are low molecular weight oligomers (5), which include noncovalently linked species (7-9) and dityrosine cross-linked ␤-amyloid protein species (CAPS) (10).Active vaccination with A␤ or passive administration of anti-A␤ antibodies has shown promise in transgenic AD animal models and in some AD patients by inducing neuritic plaque clearance, neutralizing neurotoxic soluble A␤ oligomers, and/or improving cognitive functioning (6,8,(11)(12)(13). Immunotherapy has generally targeted linear sequence epitopes on A␤ (13). However, antibodies that do not distinguish between pathologic A␤ conformers and the monomeric peptide may have adverse effects because the native peptide has been implicated in normal lipid and cholesterol homeostasis (14). Of potentially greater use are antibodies that cross-react with conformational epitopes on pathogenic aggregated A...

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Monoclonal antibodies from a patient with anti‐NMDA receptor encephalitis

Al-Saleem

Panzer

Lee

et al. 2018

Ann Clin Transl Neurol

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ObjectiveAnti‐NMDA receptor encephalitis (ANRE) is a potentially lethal encephalitis attributed to autoantibodies against the N‐methyl‐D‐aspartate receptor (NMDAR). We sought to clone and characterize monoclonal antibodies (mAbs) from an ANRE patient.MethodsWe used a hybridoma method to clone two IgG mAbs from a female patient with ANRE without teratoma, and characterized their binding activities on NMDAR‐transfected cell lines, cultured primary rat neurons, and mouse hippocampus. We also assessed their effects on voluntary locomotor activity in mice and binding to NMDAR in vivo.ResultsThe mAbs are structurally distinct and arose from distinct B‐cell lineages. They recognize different epitopes on the GluN1 amino terminal domain (ATD), yet both require amino acids important for post‐translational modification. Both mAbs bind subsets of GluN1 on cultured rat hippocampal neurons. The 5F5 mAb binds mouse brain hippocampal tissues, and the GluN1 recognized on cultured rat neurons was substantially extra‐synaptic. Antibody binding to primary hippocampal neurons induced receptor internalization. The NMDAR inhibitor MK‐801 inhibited internalization without preventing mAb binding; AP5 inhibited both mAb binding and internalization. Exposure of mice to the mAbs following permeabilization of the blood brain barrier increased voluntary wheel running activity, similar to low doses of the NMDAR inhibitor, MK‐801.InterpretationThese mAbs recapitulate features demonstrated in previous studies of ANRE patient CSF, and exert effects on NMDAR in vitro and in vivo consistent with modulation of NMDAR activity.

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Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins

Cited by 30 publications

References 45 publications

Efficient Serum Clearance of Botulinum Neurotoxin Achieved Using a Pool of Small Antitoxin Binding Agents

Efficient Serum Clearance of Botulinum Neurotoxin Achieved Using a Pool of Small Antitoxin Binding Agents

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

Monoclonal antibodies from a patient with anti‐NMDA receptor encephalitis

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