Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

Abstract

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.

Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B

“…The exchange of the dissimilar AA and very dissimilar AAs in the heavy chain of SEM120-IIIC1 led to a reduction of antigen binding. The effect of the AAs which were not exchanged in hu8SEM120-IIIC1 were observed by single back-mutations and tested by ELISA neutralization in the mouse phrenic nerve-hemdiaphragm ex vivo studies [24]. The mutation of leucine to valine in FR1 (V21>L) resulted in reduction of toxin neutralization efficiency (data not shown) compared to hu8SEM120-IIIC1 which is in accordance with the structure model.…”

DNA vaccines targeting heavy chain C-terminal fragments ofClostridium botulinumneurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge

“…Therefore, there is a need to proactively develop new therapies and vaccination strategies that can prevent intoxication. Although multiple botulinum toxin vaccines are currently in development, [25][26][27][28] the safety, immunogenicity, and stability of DNA make this vaccine platform an additional option to specifically target botulinum antigens and allow for stable delivery anywhere a bioterrorist attack may occur. In addition, the use of DNA vaccines targeting the H C of botulinum neurotoxin serotypes A, B, and E has shown positive neutralization results.…”

Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy

“…For the isolation of high-affinity antibodies against specific targets, animals are immunized with toxoids, non-toxic subunits or selected toxin domains. Alternatively, human material from immunized patients can be used for construction of immune libraries [17,79,96,97].…”

“…So far, antibodyphage display was successfully used for antibody selection against a panel of toxins classified as category A agents, such as from Clostridium botulinum (botulism) [16,25,53,79] and Bacillus anthracis (anthrax) [97] and also against different category B agents, such as staphylococcal enterotoxin B [66,110] and ricin toxin from Ricinus communis [6,96]. An example for a high-risk microorganism that produces the most toxic substances known with the highest risk of potential use as bioweapons is the Gram-positive, anaerobic, spore-forming bacterium Clostridium botulinum and other Clostridium subspecies.…”

“…Here, antibody phage display provides a technology to generate toxin-neutralizing antibodies against each serotype. For instance, a macaque immune library was used to isolate neutralizing scFv with nm affinities against the light chain of BoNT/A [16,79], but also antibodies against the heavy chain or other relevant serotypes of BoNT are of therapeutical interest. Phage display technology was also used for isolation of single domain antibodies (VHH) after immunization of a llama with a cocktail of seven BoNT toxoids (A-F) [25].…”