Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library.
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.
Technische Universität Braunschweig; Institut für Biochemie, Biotechnologie, und Bioinformatik; Abteilung Biotechnologie; Braunschweig, Germany.
Sebastian
S
Miethe
National Institute for Biological Standards and Control (NIBSC); Medicines and Healthcare Products Regulatory Agency; Division of Bacteriology; Potters Bar, UK.
Christine
C
Rasetti-Escargueil
National Institute for Biological Standards and Control (NIBSC); Medicines and Healthcare Products Regulatory Agency; Division of Bacteriology; Potters Bar, UK.
Yvonne
Y
Liu
Institut de Recherche Biomédicale des Armées (IRBA-CRSSA); Département de Microbiologie; Unité de biotechnologie des anticorps et des toxines ; La Tronche Cedex, France.
Siham
S
Chahboun
Institut de Recherche Biomédicale des Armées (IRBA-CRSSA); Département de Microbiologie; Unité de biotechnologie des anticorps et des toxines ; La Tronche Cedex, France.
Thibaut
T
Pelat
Institut de Recherche Biomédicale des Armées (IRBA-CRSSA); Département de Microbiologie; Unité de biotechnologie des anticorps et des toxines ; La Tronche Cedex, France.
Arnaud
A
Avril
Technische Universität Braunschweig; Institut für Biochemie, Biotechnologie, und Bioinformatik; Abteilung Biotechnologie; Braunschweig, Germany.
André
A
Frenzel
Technische Universität Braunschweig; Institut für Biochemie, Biotechnologie, und Bioinformatik; Abteilung Biotechnologie; Braunschweig, Germany.
Thomas
T
Schirrmann
Institut de Recherche Biomédicale des Armées (IRBA-CRSSA); Département de Microbiologie; Unité de biotechnologie des anticorps et des toxines ; La Tronche Cedex, France.
Philippe
P
Thullier
National Institute for Biological Standards and Control (NIBSC); Medicines and Healthcare Products Regulatory Agency; Division of Bacteriology; Potters Bar, UK.
Dorothea
D
Sesardic
Technische Universität Braunschweig; Institut für Biochemie, Biotechnologie, und Bioinformatik; Abteilung Biotechnologie; Braunschweig, Germany.
Michael
M
Hust
D000818:Animals; D019138:Antibodies, Blocking / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000009:adverse effects / Q000276:immunology*; D001906:Botulism / Q000150:complications / Q000276:immunology / Q000628:therapy*; D061505:Cell Surface Display Techniques; D047013:Clostridium botulinum type A / Q000276:immunology*; D018604:Epitope Mapping; D006801:Humans; D007109:Immunity / Q000235:genetics; D007114:Immunization; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D054444:Immunoglobulin Light Chains, Surrogate / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D007167:Immunotherapy / Q000379:methods*; D008252:Macaca fascicularis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010243:Paralysis / Q000209:etiology / Q000276:immunology / Q000517:prevention & control*; D010791:Phrenic Nerve / Q000187:drug effects* / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*
D019138:Antibodies, Blocking; D007141:Immunoglobulin Fc Fragments; D054444:Immunoglobulin Light Chains, Surrogate; D057127:Single-Chain Antibodies; D019274:Botulinum Toxins, Type A
BoNT/A; Clostridium botulinum; Macaca fascicularis; SNAP-25; botulinum neurotoxin; ex-vivo assay; in vitro assay; macaque; mouse phrenic nerve-hemidiaphragm assay; phage display; scFv; scFv-Fc