In vivo neutralization of α-Cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody

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DOIResolve DOI: http://doi.org/10.1371/journal.pone.0069495
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TypeArticle
Journal titlePLoS ONE
ISSN1932-6203
Volume8
Issue7
Article numbere69495
Subjectantibody; cobrotoxin; single variable domain antibody; single variable domain Fc antibody; snake venom antiserum; unclassified drug; animal cell; animal experiment; animal model; antibody affinity; article; controlled study; drug efficacy; drug penetration; envenomation; female; humoral immunity; in vivo study; lethality; male; molecular weight; mouse; Nicotiana benthamiana; nonhuman; protein expression; protein purification
AbstractSmall recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab')2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α-Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α-Cbtx. Mouse α-Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α-Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. © 2013 Richard et al.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); Human Health Therapeutics (HHT-TSH)
Peer reviewedYes
NPARC number21269926
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Record identifier2684e8b0-3433-4c7d-b8ff-e86f85e0ad0d
Record created2013-12-13
Record modified2016-05-09
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