The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.Peer reviewe

Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.Peer reviewe

Antibodies Against Anthrax: Mechanisms of Action and Clinical Applications

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

Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

Background 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. Results 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. Conclusion 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.Peer reviewe

Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required

Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus

Obtention et ingénierie d'anticorps recombinants thérapeutiques et/ou prophylactiques dirigés contre les agents du risque biologique provoqué

Les anticorps recombinants (Ac) de primates non humains (PNH) représentent une approche très prometteuse en vue de la prise en charge médicale d'agents du risque biologique provoqué ( armes biologiques ). Nous avons montré les avantages de cette approche et ses particularités méthodologiques. Un scFv neutralisant la toxine létale de B. anthracis (2LF : KD= 1,02 nM), et dirigé contre la sous-unité facteur létal , a été isolé à partir d'une banque immune de scFv de PNH exposée à la surface de phages, et caractérisé. Un autre scFv, 43RCA, neutralisant la ricine, a été isolé par la même technique puis évalué (en particulier : KD= 40 pM). Cette approche peut aussi s'appliquer à des pathogènes n'appartenant pas au risque biologique provoqué, puisqu'un scFv dirigé contre Aspergillus fumigatus a aussi été isolé (MS130i-IIIC3 : KD= 0,96 nM). Les outils d'analyse de séquences disponibles en ligne sur le site IMGT® ont permis de montrer la grande similarité de ces scFv avec leurs homologues humains. Des travaux d'ingénierie de fragments d'Ac ont aussi été réalisés, dont une maturation d'affinité in vitro (KD initial = 3,4 nM ; KD final = 0,18 nM). Une approche particulière de l'humanisation ("germline humanization") utilisant les régions charpentes (FR) d'IgM (rencontrés chez tous les sujets à la différence des FR d'IgG) et la standardisation IMGT® a été implémentée pour assurer une tolérance optimale de l'un de nos scFv. Des essais in vivo d'une IgG primatisée ont démontré ses capacités prophylactique et thérapeutiqueRecombinant antibodies isolated from non-human primates represent very promising medical countermeasures against bioweapons. The advantages and methodological aspects of this approach have been described. An scFv, neutralizing the lethal toxin of Bacillus anthracis (2LF: KD= 1.02 nM) and directed against the lethal factor subunit was isolated from a phage-displayed immune library, and characterised. Another scFv, 43RCA, neutralizing ricin, was obtained with the same methodology and tested (in particular: KD= 40 pM). This approach may also be used beyond bioweapons, as an scFv directed toward Aspergillus fumigatus was also isolated (MS130i-IIIC3: KD= 0,96 nM). On-line sequence analysis with IMGT tools allowed to show the high degree of similarity between these scFvs and their human counterparts. Antibody fragments were engineered, including an in vitro affinity maturation (KD initial = 3.4 nM; KD final = 0.18 nM). Utilizing IMGT standardisation and on-line tools, a germline humanization - utilizing FR derived from IgM, encountered by every Human, as opposed to IgG FR - was realized in order to ensure an optimal tolerance for one of our scFvs. A primatized IgG was tested in vivo and showed therapeutic and prophylactic capacitiesMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF