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PubMed | Institute Of Recherche Biomedicale Des Armees Irba Crssa, Directorate General of Armaments, New York University, LFB Biotechnologies and 7 more.
Type: | Journal: Journal of controlled release : official journal of the Controlled Release Society | Year: 2016

The high toxicity of ricin and its ease of production have made it a major bioterrorism threat worldwide. There is however no efficient and approved treatment for poisoning by ricin inhalation, although there have been major improvements in diagnosis and therapeutic strategies. We describe the development of an anti-ricin neutralizing monoclonal antibody (IgG 43RCA-G1) and a device for its rapid and effective delivery into the lungs for an application in humans. The antibody is a full-length IgG and binds to the ricin A-chain subunit with a high affinity (KD=53pM). Local administration of the antibody into the respiratory tract of mice 6h after pulmonary ricin intoxication allowed the rescue of 100% of intoxicated animals. Specific operational constraints and aerosolization stresses, resulting in protein aggregation and loss of activity, were overcome by formulating the drug as a dry-powder that is solubilized extemporaneously in a stabilizing solution to be nebulized. Inhalation studies in mice showed that this formulation of IgG 43RCA-G1 did not induce pulmonary inflammation. A mesh nebulizer was customized to improve IgG 43RCA-G1 deposition into the alveolar region of human lungs, where ricin aerosol particles mostly accumulate. The drug delivery system also comprises a semi-automatic reconstitution system to facilitate its use and a specific holding chamber to maximize aerosol delivery deep into the lung. In vivo studies in monkeys showed that drug delivery with the device resulted in a high concentration of IgG 43RCA-G1 in the airways for at least 6h after local deposition, which is consistent with the therapeutic window and limited passage into the bloodstream.


Miethe S.,TU Braunschweig | Rasetti-Escargueil C.,UK National Institute for Biological Standards and Control | Liu Y.,UK National Institute for Biological Standards and Control | Chahboun S.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | And 7 more authors.
mAbs | Year: 2014

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease. © 2014 Landes Bioscience.


PubMed | Institute Of Recherche Biomedicale Des Armees Irba Crssa, Institute Pasteur Paris, University of Helsinki, UK National Institute for Biological Standards and Control and TU Braunschweig
Type: Journal Article | Journal: PloS one | Year: 2015

Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure.In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay.These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.


PubMed | Institute Of Recherche Biomedicale Des Armees Irba Crssa, UK National Institute for Biological Standards and Control and TU Braunschweig
Type: Journal Article | Journal: mAbs | Year: 2014

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.


Drouet J.-B.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Michel V.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Peinnequin A.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Alonso A.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | And 5 more authors.
Psychoneuroendocrinology | Year: 2010

Metyrapone, a cytochrome P450 inhibitor used to inhibit corticosterone synthesis, triggers biological markers of stress and also reduces stress-induced anxiety-like behaviors. To address these controversial effects, 6 separate investigations were carried out. In a first set of investigations, abdominal temperature (Tabd), spontaneous locomotor activity (AS) and electroencephalogram (EEG) were recorded in freely moving rats treated with either saline or 150mgkg-1 metyrapone. An increase in Tabd and AS occurred in saline rats, while, metyrapone rats exhibited an immediate decrease, both variables returning to basal values 5h later. Concomitantly, the EEG spectral power increased in the gamma and beta 2 bands and decreased in the alpha frequency band, and the EMG spectral power increased. This finding suggests that metyrapone depressed stress-induced physiological response while arousing the animal. In a second step, restraint stress was applied 5h after injection. Metyrapone significantly blunted the stress-induced Tabd and AS rise, without affecting the brain c-fos mRNA increase. Corticosterone (5 and 40mgkg-1) injected concomitantly to metyrapone failed to reverse the observed metyrapone-induced effects in Tabd and AS. Finasteride (50mgkg-1), which blocks neurosteroid production, was also unable to block these effects. In conclusion, metyrapone acutely reduced stress-induced physiological response in freely behaving rats independently from glucocorticoids and neurosteroids. © 2010 Elsevier Ltd.


Froude II J.W.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Froude II J.W.,U.S. Army | Thullier P.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Pelat T.,Institute Of Recherche Biomedicale Des Armees Irba Crssa
Toxins | Year: 2011

B. anthracis is a bioweapon of primary importance and its pathogenicity depends on its lethal and edema toxins, which belong to the A-B model of bacterial toxins, and on its capsule. These toxins are secreted early in the course of the anthrax disease and for this reason antibiotics must be administered early, in addition to other limitations. Antibodies (Abs) may however neutralize those toxins and target this capsule to improve anthrax treatment, and many Abs have been developed in that perspective. These Abs act at various steps of the cell intoxication and their mechanisms of action are detailed in the present review, presented in correlation with structural and functional data. The potential for clinical application is discussed for Abs targeting each step of entry, with four of these molecules already advancing to clinical trials. Paradoxically, certain Abs may also enhance the lethal toxin activity and this aspect will also be presented. The unique paradigm of Abs neutralizing anthrax toxins thus exemplifies how they may act to neutralize A-B toxins and, more generally, be active against infectious diseases. © 2011 by the authors; licensee MDPI, Basel, Switzerland.


Froude J.W.,U.S. Army | Froude J.W.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Stiles B.,U.S. Army | Pelat T.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Thullier P.,Institute Of Recherche Biomedicale Des Armees Irba Crssa
mAbs | Year: 2011

Potential bioweapons are biological agents (bacteria, viruses and toxins) at risk of intentional dissemination. Biodefense, defined as development of therapeutics and vaccines against these agents, has seen an increase, particularly in the US, following the 2001 anthrax attack. This review focuses on recombinant antibodies and polyclonal antibodies for biodefense that have been accepted for clinical use. These antibodies aim to protect against primary potential bioweapons or category A agents as defined by the Centers for Disease Control and Prevention (Bacillus anthracis, Yersinia pestis, Francisella tularensis, botulinum neurotoxins, smallpox virus and certain others causing viral hemorrhagic fevers) and certain category B agents. Potential for prophylactic use is presented, as well as frequent use of oligoclonal antibodies or synergistic effect with other molecules. Capacities and limitations of antibodies for use in biodefense are discussed, and are generally applicable to the field of infectious diseases. © 2011 Landes Bioscience.


Chahboun S.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Hust M.,TU Braunschweig | Liu Y.,Public Health England | Pelat T.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | And 5 more authors.
BMC Biotechnology | Year: 2011

Background: Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use.Results: Diversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screening with several rounds of increasing stringency, as is usual with phage technology, the library got overwhelmed by phagemids encoding incomplete scFvs. The screening was successfully re-performed with a single round of high stringency. In particular, one of the isolated scFvs, 2H8, bound BoNT/A1 with a 3.3 nM affinity and effectively inhibited BoNT/A1 endopeptidase activity. The sequence encoding 2H8 was 88% identical to human germline genes and its average G-score was -0.72, quantifying the high human-like quality of 2H8.Conclusions: The presence of restrictions sites within many of the sequences that were to be part of the library did not prevent the isolation of an scFv, 2H8, by an adapted panning strategy. ScFv 2H8 inhibited toxin endopeptidase activity in vitro and possessed human-like quality required for clinical development. More generally, the construction and screening of phage-displayed libraries built from hyper-immunized non-human primates is an efficient solution to isolate antibody fragments with therapeutic potential. © 2011 Chahboun et al; licensee BioMed Central Ltd.


Hulseweh B.,Wehrwissenschaftliches Institute For Schutztechnologien Wis | Rulker T.,TU Braunschweig | Pelat T.,Institute Of Recherche Biomedicale Des Armees Irba Crssa | Langermann C.,Wehrwissenschaftliches Institute For Schutztechnologien Wis | And 5 more authors.
mAbs | Year: 2014

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69-3A2) neutralized 50% of 5 x 104 TCID50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69-3A2 antibody is a promising candidate for future passive vaccine development. © 2014 Landes Bioscience.


PubMed | Institute Of Recherche Biomedicale Des Armees Irba Crssa, Ecole Polytechnique Federale de Lausanne and TU Braunschweig
Type: | Journal: BMC biotechnology | Year: 2015

Beside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimers disease (AD) being composed of aggregated fibrils of -amyloid (A). During the underlying fibrillogenic pathway, starting from a surplus of soluble A and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of -amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of A.Here we report the generation and characterization of a variety of amyloid- specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from A42 peptides. These antigens were used for the generation of two A42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two nave human phage display libraries resulted in multiple unique binders specific for amyloid-. Three of the obtained antibodies target the N-terminal part of A42 although with varying epitopes, while another scFv binds to the -helical central region of the peptide. The affinities of the antibodies to various A42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis.We demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti -amyloid scFvs that are capable of accurately discriminating between minute conformational differences.

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