A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

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

A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A.

The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination

Chemical interactions of woodsmoke constituents and food components.

Model systems were used to investigate certain aspects of the chemical reactions which occur when proteinaceous foods are smoked, especially the role of aldehydes in smoke and basic moieties of the foods. The studies focused upon possible imine formation between aldehydes and free amino acids or lysine side chains in the proteins. Imines were chosen for investigation since the reported characteristics of smoked foods, such as colour, texture and reduction in nutritional value could be due to imine formation. By performing chromatographic (HPLC, TLC) , spectroscopic (UV-vis., IR) and electrophoretic (SDS PAGE) experiments with model systems containing amino acids or proteins, the formation of imines was indicated. Furthermore, other non-imine products were indicated, specifically, cyclization products of histidine and tryptophan. Non-imine products from the 1,2-dihydroxybenzene components of woodsmoke with methionine were also indicated. In further studies the binding affinity of various smoke constituents for fish protein was investigated, however, no differences were detected between aldenydic and phenolic components with respect to the rate of their extraction from fish. It is suggested that this was due to the lability of imines in aqueous solution

Chemical interactions of woodsmoke constituents and food components.

Model systems were used to investigate certain aspects of the chemical reactions which occur when proteinaceous foods are smoked, especially the role of aldehydes in smoke and basic moieties of the foods. The studies focused upon possible imine formation between aldehydes and free amino acids or lysine side chains in the proteins. Imines were chosen for investigation since the reported characteristics of smoked foods, such as colour, texture and reduction in nutritional value could be due to imine formation. By performing chromatographic (HPLC, TLC) , spectroscopic (UV-vis., IR) and electrophoretic (SDS PAGE) experiments with model systems containing amino acids or proteins, the formation of imines was indicated. Furthermore, other non-imine products were indicated, specifically, cyclization products of histidine and tryptophan. Non-imine products from the 1,2-dihydroxybenzene components of woodsmoke with methionine were also indicated. In further studies the binding affinity of various smoke constituents for fish protein was investigated, however, no differences were detected between aldenydic and phenolic components with respect to the rate of their extraction from fish. It is suggested that this was due to the lability of imines in aqueous solution

Solution structure of &#969;-conotoxin MVIIC, a high affinity ligand of P-type calcium channels, using <SUP>1</SUP>H NMR spectroscopy and complete relaxation matrix analysis

We have determined the solution structure of the ω-conotoxin MVIIC fromConus magusby 1H NMR. This conopeptide preferentially blocks P and Q type Ca2+ currents by binding with high affinity to voltage-sensitive Ca2+ channels in neurons. This 26 residue peptide with three disulfide bonds was chemically synthesized and refolded for NMR structural studies. The 1H NMR NOESY spectrum of this peptide was completely assigned, with stereospecific assignments made for 12 of the β prochiral centers. Complete relaxation matrix analysis using MARDIGRAS was used to obtain initial interproton distances from peak intensities. The correlation time necessary for these calculations was determined by measuring 13C relaxation times using inversely detected natural abundance spectra. Distances were input to DG, which provided 15 starting structures which were then subjected to restrained molecular dynamics calculations using SANDER with the AMBER 91 force fieldin vacuo. 1H-1H vicinal coupling constants were obtained using a combination of line fitting of both E. COSY and NOESY spectra and used to generate angle restraints that were included explicitly in the restrained molecular dynamics calculations. The final set of the 15 best structures had a backbone rmsd of 0.84 Å. The ensemble R⅙ factor calculated by CORMA for the final 15 structures was 11%. The final structure consists of an anti-parallel, triple-stranded β-sheet, with four turns. In spite of significant differences in amino acid sequence and affinities for calcium channel subtypes, the backbone structure of ω-conotoxin MVIIC is very similar to the previously reported structure of ω-conotoxin GVIA

An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E.

Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration

Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT/F.

Generating specific monoclonal antibodies (mAbs) that neutralize multiple antigen variants is challenging. Here, we present a strategy to generate mAbs that bind seven subtypes of botulinum neurotoxin serotype F (BoNT/F) that differ from each other in amino acid sequence by up to 36%. Previously, we identified 28H4, a mouse mAb with poor cross-reactivity to BoNT/F1, F3, F4, and F6 and with no detectable binding to BoNT/F2, F5, or F7. Using multicolor labeling of the different BoNT/F subtypes and fluorescence-activated cell sorting (FACS) of yeast displayed single-chain Fv (scFv) mutant libraries, 28H4 was evolved to a humanized mAb hu6F15.4 that bound each of seven BoNT/F subtypes with high affinity (KD 5.81 pM to 659.78 pM). In contrast, using single antigen FACS sorting, affinity was increased to the subtype used for sorting but with a decrease in affinity for other subtypes. None of the mAb variants showed any binding to other BoNT serotypes or to HEK293 or CHO cell lysates by flow cytometry, thus demonstrating stringent BoNT/F specificity. Multicolor FACS-mediated antibody library screening is thus proposed as a general method to generate multi-specific antibodies to protein subtypes such as toxins or species variants

DSC thermograms of anti-BoNT 9ABE and mAbs comprising anti-BoNT 9ABE in the succinate, arginine, Polysorbate formulation, ramp rate 1°C/minute.

<p>Results are from single samples. <b>A.</b> Thermograms of individual mAbs compared to anti-BoNT 9ABE. Individual mAbs are: mAb A-a, -b, -c, which bind BoNT/A; mAb B-a, -b, -c, which bind BoNT/B; and mAb E-a, E-b, E-c, which bind BoNT/E. <b>B</b>. the amplified anti-BoNT 9ABE thermogram overlaid with those of mAbs with the lowest and highest Tm’s–mAb A-b and mAb B-a respectively. Panels C through E compare measured and calculated DSC thermograms normalized for concentration. <b>C.</b> Three mAbs that bind to BoNT/B; <b>D.</b> Three mAbs that bind BoNT/E. <b>E.</b> DSC thermograms of nine-antibody mixture (anti-BoNT 9ABE measured) compared to the sum calculated from the measured thermograms of the nine individual antibodies (anti-BoNT 9ABE calculated) and normalized for concentration. Also shown are thermograms for three mAbs binding BoNT/A (3A), BoNT/B (3B) and BoNT/E (3E).</p