Abstract
Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.
Highlights
Botulinum type E neurotoxins (BoNT/E) belong to one of at least seven immunologically distinct groups of neurotoxins (BoNT/A-G and botulinum neurotoxin (BoNT)/HA [1,2]) produced by different species of bacteria from the genus Clostridium [3,4]
Yeast-displayed single chain Fv antibody libraries were constructed from the VH and Vk genes of human volunteers immunized with pentavalent BoNT toxoid
Yeast display and sorting of single chain Fv (scFv) libraries constructed from the V-genes of humans immunized with BoNT toxoid was used to isolate a panel of ten unique human monoclonal antibody (mAb) to BoNT/E
Summary
Botulinum type E neurotoxins (BoNT/E) belong to one of at least seven immunologically distinct groups of neurotoxins (BoNT/A-G and BoNT/HA [1,2]) produced by different species of bacteria from the genus Clostridium [3,4]. E5 [7,8]) and Clostridium botulinum (subtypes E1, E2, E3 [9] E6 [10], E7, E8 [11], E9 [12], E10, E11 [13], and E12 [14]). BoNTs are produced as a single polypeptide. In order to reach full catalytic activity, the progenitor BoNT polypeptide is cleaved between the proteolytic domain and the translocation domain, with the two resulting peptides being linked by a disulfide bridge. Toxins produced in proteolytic C. botulinum strains are cleaved during processing, but in non-proteolytic strains, such as those that produce all type E and some type B toxins, trypsinization is used to cleave the LC-HN and achieve full toxicity [20]
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