Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

Abstract

The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT͞A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT͞A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents. monoclonal antibody ͉ immunotherapy ͉ antibody engineering ͉ vaccine ͉ phage display

Production and purification of a chimeric monoclonal antibody against botulinum neurotoxin serotype A

“…These values are also similar to those previously determined for both the S25 antibody and other antibodies [3], [34] and [35]. Table 2).…”

“…To meet the increased demand for monoclonal antibodies, all aspects of antibody production and purification need to be improved. Another potential use of monoclonal antibodies is the treatment for exposure to toxins, such as botulism neurotoxin (BoNT), one of the most poisonous substances known [3]. BoNT has been classified by the Centers of Disease Control (CDC) as one of the six highest risk threats for use in bioterrorism due to its potency, lethality, and ease of production [4].…”

“…Recombinant monoclonal antibodies are currently being developed for the treatment of botulism. Three monoclonal antibodies have been combined to neutralize 450,000 50% lethal doses of BoNT serotype A [3]. Half of the mice treated with a combination of three monoclonal antibodies were able to survive exposure to 450,000 times the amount of BoNT serotype A that would normally kill 50% of mice.…”

Enhancing the Protective Immune Response against Botulism

“…Therefore, antibodies generated to the HCR/A(W1266A) vaccine likely mark circulating BoNT/A for clearance and inhibit receptor recognition. This is consistent with the observation that BoNTs can be cleared by combinations of monoclonal antibodies that do not neutralize toxin activity (59). Furthermore, work by Atassi and his coworkers identified the peptide composing the GPB as a minor yet reproducible immunological epitope, though dominant epitopes varied by species (58).…”

Fine and Domain-level Epitope Mapping of Botulinum Neurotoxin Type A Neutralizing Antibodies by Yeast Surface Display

“…After conversion to IgG with human Fc, all three mAbs showed in vitro BoNT/A neutralization, as evidenced by a prolongation in the time to paralysis in the hemidiaphragm assay, and in vivo neutralization, as evidenced by a prolongation in time to death in mice challenged with toxin (Table 1, and ref. 17 ). For two of the mAbs, 3D12 and HuC25, fine epitope mapping was accomplished using an approach first described by Chao et al 21 by: 1) generating a library of surface displayed BoNT/A H C mutants; 2) selecting for loss of binding of the mAb being epitope mapped; 3) analyzing the location of mutations leading to loss of mAb binding; and 4) constructing single yeast displayed BoNT/A H C alanine mutants in the putative mAb epitope and measuring the energetic contribution of the toxin side chain to mAb binding (ΔΔG).…”