Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

Section: Discussionmentioning

confidence: 95%

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

Wang

Fang

et al. 2016

“…For anti-BoNT/E-HC antibodies, a titer of 1/600,000 against BoNT/E3 holotoxin was observed. Finally, for anti-BoNT/E-LC antibodies, a titer of 1/600,000 against BoNT/E3 holotoxin was observed [ 51 , 73 , 74 , 75 ].…”

Section: Animal Immunization and Antibody Phage-display Library Comentioning

confidence: 99%

“…The immune-library directed against BoNT/B2-HC consisted of 1 × 10 8 independent clones and was finally packaged in M13K07 helper-phage. The immune-library directed against BoNT/B2-LC consisted of 1.1 × 10 8 unique clones and was finally packaged in M13K07 phage [ 74 ]. The immune-library directed against BoNT/E3-LC consisted of 9.6 × 10 8 independent clones with a full-size insert rate of 88%.…”

Section: Animal Immunization and Antibody Phage-display Library Comentioning

confidence: 99%

“…Ten different scFvs were identified, expressed as soluble scFvs, purified and their affinities for BoNT/B1 and BoNT/B2-LC were measured. The multistep panning approach was validated because the number of eluted phage was comparable to that reported in previous studies and sub-nanomolar scFvs were produced [ 1 , 74 ].…”

Section: Animal Immunization and Antibody Phage-display Library Comentioning

confidence: 99%

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The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

Rasetti-Escargueil

Avril²,

Miethe

et al. 2017

Self Cite

The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.

“…An antibody directed against BoNT/E LC, which was selected using the biochemical assay (based on in vitro inhibition of the endopeptidase activity), proved to have a highly neutralizing potency in vivo [ 13 ]. Similarly, antibodies against BoNT/A and BoNT/B LCs, which were also selected by in vitro assays [ 22 ], were neutralizing in vivo when presented in combination with antibodies against the HC of the corresponding toxin serotype [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

Bak

Rajagopal

Stickings

et al. 2017

Self Cite

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.