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

Fan Y, Dong J, Lou J, Wen W, Conrad F, Geren IN, Garcia-Rodriguez C, Smith TJ, Smith LA, Ho M, Pires-Alves M, Wilson BA, Marks JD - Toxins (Basel) (2015)

Bottom Line: Eleven mAbs inhibited BoNT/B LC proteolytic activity.The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site.The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. frank.fan@ucsf.edu.

ABSTRACT
Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

No MeSH data available.


Related in: MedlinePlus

Cartoon of mAb epitope clusters. mAbs were clustered based on their ability to simultaneously bind BoNT/B LC with one of the scFv displayed on the surface of yeast used to capture BoNT/B LC out of solution and then the ability of a second scFv to bind the captured B LC determined. mAb epitopes are shown as circles; overlapping circles indicate mAb pairs that cannot simultaneously bind LC. Red circles indicate mAbs that inhibited BoNT/ B endopeptidase activity; green circles represent non-inhibitory mAbs. Dotted circles indicate human-derived mAbs, and solid circles mouse-derived mAbs. Epitope cluster 1 antibodies were sub-divided into three groups based on degree of inhibition of BoNT/ B endopeptidase activity.
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toxins-07-03405-f001: Cartoon of mAb epitope clusters. mAbs were clustered based on their ability to simultaneously bind BoNT/B LC with one of the scFv displayed on the surface of yeast used to capture BoNT/B LC out of solution and then the ability of a second scFv to bind the captured B LC determined. mAb epitopes are shown as circles; overlapping circles indicate mAb pairs that cannot simultaneously bind LC. Red circles indicate mAbs that inhibited BoNT/ B endopeptidase activity; green circles represent non-inhibitory mAbs. Dotted circles indicate human-derived mAbs, and solid circles mouse-derived mAbs. Epitope cluster 1 antibodies were sub-divided into three groups based on degree of inhibition of BoNT/ B endopeptidase activity.

Mentions: Nineteen mAbs with unique VH CDR3 binding BoNT/B LC were isolated from the human and mouse libraries, as determined by DNA sequencing (Table 1 and Table 2). Equilibrium binding constants (KD) for yeast-displayed scFv binding to BoNT/B LC ranged from 0.24–14.3 nM with an average KD of 3.27 nM (Table 2). The epitopes recognized by each scFv were classified into epitope groups based on their ability to compete with each other for binding to BoNT/B LC. In the assay, BoNT/B1 LC (or holotoxins of other subserotypes) captured by yeast-displayed scFv was probed with E. coli-expressed soluble scFv. The 19 mAbs were grouped into three epitope clusters (I-III) based on their ability to compete for LC binding (Figure 1). The largest cluster (I) was shared by 13 scFvs, at least one of each overlapped with other cluster I mAbs. Given the large number of mAbs, the epitope cluster was further divided into three subgroups (I-1, I-2 and I-3) based on the degree of inhibition.


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

Fan Y, Dong J, Lou J, Wen W, Conrad F, Geren IN, Garcia-Rodriguez C, Smith TJ, Smith LA, Ho M, Pires-Alves M, Wilson BA, Marks JD - Toxins (Basel) (2015)

Cartoon of mAb epitope clusters. mAbs were clustered based on their ability to simultaneously bind BoNT/B LC with one of the scFv displayed on the surface of yeast used to capture BoNT/B LC out of solution and then the ability of a second scFv to bind the captured B LC determined. mAb epitopes are shown as circles; overlapping circles indicate mAb pairs that cannot simultaneously bind LC. Red circles indicate mAbs that inhibited BoNT/ B endopeptidase activity; green circles represent non-inhibitory mAbs. Dotted circles indicate human-derived mAbs, and solid circles mouse-derived mAbs. Epitope cluster 1 antibodies were sub-divided into three groups based on degree of inhibition of BoNT/ B endopeptidase activity.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4591640&req=5

toxins-07-03405-f001: Cartoon of mAb epitope clusters. mAbs were clustered based on their ability to simultaneously bind BoNT/B LC with one of the scFv displayed on the surface of yeast used to capture BoNT/B LC out of solution and then the ability of a second scFv to bind the captured B LC determined. mAb epitopes are shown as circles; overlapping circles indicate mAb pairs that cannot simultaneously bind LC. Red circles indicate mAbs that inhibited BoNT/ B endopeptidase activity; green circles represent non-inhibitory mAbs. Dotted circles indicate human-derived mAbs, and solid circles mouse-derived mAbs. Epitope cluster 1 antibodies were sub-divided into three groups based on degree of inhibition of BoNT/ B endopeptidase activity.
Mentions: Nineteen mAbs with unique VH CDR3 binding BoNT/B LC were isolated from the human and mouse libraries, as determined by DNA sequencing (Table 1 and Table 2). Equilibrium binding constants (KD) for yeast-displayed scFv binding to BoNT/B LC ranged from 0.24–14.3 nM with an average KD of 3.27 nM (Table 2). The epitopes recognized by each scFv were classified into epitope groups based on their ability to compete with each other for binding to BoNT/B LC. In the assay, BoNT/B1 LC (or holotoxins of other subserotypes) captured by yeast-displayed scFv was probed with E. coli-expressed soluble scFv. The 19 mAbs were grouped into three epitope clusters (I-III) based on their ability to compete for LC binding (Figure 1). The largest cluster (I) was shared by 13 scFvs, at least one of each overlapped with other cluster I mAbs. Given the large number of mAbs, the epitope cluster was further divided into three subgroups (I-1, I-2 and I-3) based on the degree of inhibition.

Bottom Line: Eleven mAbs inhibited BoNT/B LC proteolytic activity.The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site.The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. frank.fan@ucsf.edu.

ABSTRACT
Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

No MeSH data available.


Related in: MedlinePlus