Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

Abstract

The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

“…We previously reported that the antibodies that most potently inhibited BoNT/A are bound at the alpha-exosite, where the α-helix of substrate SNAP-25 binds, and are remote from the substrate cleavage site [ 28 , 29 ]. In contrast, the mAbs showing the greatest inhibition of BoNT/B activity bound at the catalytic site with the degree of inhibition decreasing with distance of the epitope from the catalytic site.…”

“…We previously reported the isolation of a single-domain camelid VHH antibody that bound the BoNT/A LC alpha exosite with a K D of 147 pM and potently inhibited SNAP25 cleavage [ 28 ]. More recently we have reported scFv and IgG mAbs that bind BoNT/A LC and inhibit SNAP25 cleavage, and like the VHH, these inhibitory mAbs bind at the alpha exosite [ 29 ]. Here, we report generation of mouse and fully human antibodies that can inhibit BoNT/B LC proteolytic activity, as well as identification of the mAb epitopes mediating this inhibition.…”

The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA

“…The BoNT/H LC-H N scFv yeast display library was constructed as described [31]. BoNT/H specific binders were isolated using three rounds of FACS sorting in which 100 nM of BoNT/H LC-H N was used for yeast incubation, followed by Alexa-647-conjugated 6F5.4 IgG (6F5.4-647) for labeling.…”

“…In separate experiments, the BoNT/H LC-H N immunized library was also stained with 100 nM of BoNT/F5 LC-H N followed by incubation with mAb 6F5.4-647 to isolate BoNT/F5 specific mAbs. Individual colonies were picked from SD-CAA plates, cultured and scFv display induced for further characterization [31]. Libraries made from mice immunized with BoNT/F fragments [18] were sorted as described above (Table 1).…”

Picolinic acids as β-exosite inhibitors of botulinum neurotoxin A light chain

“…12–17 Despite promising preclinical data, none of these compounds have progressed to clinical trials, begging the need for alternative LC inhibition strategies at other sites on the enzyme; preliminary reports of α- and β-exosite inhibition have shown promise. 18–21 …”