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

View Article: PubMed Central - PubMed

ABSTRACT

Tetanus neurotoxin (TeNT) produced by Clostridiumtetani 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.

No MeSH data available.


The 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (reducing) of purified 2-7G, 2-2D, and S-4-7H IgG4.
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toxins-08-00266-f003: The 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (reducing) of purified 2-7G, 2-2D, and S-4-7H IgG4.

Mentions: Due to the rapid clearance of scFv from serum, in vivo toxin neutralization activity might not be determined precisely. To establish more stable antibodies, 2-7G, 2-2D, and S-4-7H IgG4 were constructed by inserting the VH and VL genes into the mammalian expression vectors H293 and L293-C λ, resulting in the fusion of VH and VL genes to CH and CL genes. IgG4 antibodies could barely activate the classic pathway of complement, and thus the neutralizing antibodies could not be cleared immediately by macrophages in vivo. The plasmids were transfected into FreeStyleTM 293-F cells for instantaneous expression. The FreeStyleTM 293 Expression System was designed to allow for the large-scale transfection of suspension 293 human embryonic kidney cells in a defined, serum-free medium. The IgG4 antibodies of 2-7G, 2-2D, and S-4-7H yielded high expression levels in this system, and were purified on a Protein A column. The purified protein contained a 50-kDa heavy chain and a 28-kDa light chain, which corresponded with the entire antibody (Figure 3). Since IgGs were glycosylated and scFv from E. coli not, we measured the three IgGs with TeNT by SPR. The antigen binding affinity of 2-7G, 2-2D, and S-4-7H of the IgG was significantly higher (lower KD) than for the corresponding scFv. 2-7G, 2-2D, and S-4-7H antibodies with a KD of 5.20 × 10−11 M, 1.12 × 10−9 M and 3.57 × 10−10 M were obtained (Table 4).


Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library
The 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (reducing) of purified 2-7G, 2-2D, and S-4-7H IgG4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037492&req=5

toxins-08-00266-f003: The 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (reducing) of purified 2-7G, 2-2D, and S-4-7H IgG4.
Mentions: Due to the rapid clearance of scFv from serum, in vivo toxin neutralization activity might not be determined precisely. To establish more stable antibodies, 2-7G, 2-2D, and S-4-7H IgG4 were constructed by inserting the VH and VL genes into the mammalian expression vectors H293 and L293-C λ, resulting in the fusion of VH and VL genes to CH and CL genes. IgG4 antibodies could barely activate the classic pathway of complement, and thus the neutralizing antibodies could not be cleared immediately by macrophages in vivo. The plasmids were transfected into FreeStyleTM 293-F cells for instantaneous expression. The FreeStyleTM 293 Expression System was designed to allow for the large-scale transfection of suspension 293 human embryonic kidney cells in a defined, serum-free medium. The IgG4 antibodies of 2-7G, 2-2D, and S-4-7H yielded high expression levels in this system, and were purified on a Protein A column. The purified protein contained a 50-kDa heavy chain and a 28-kDa light chain, which corresponded with the entire antibody (Figure 3). Since IgGs were glycosylated and scFv from E. coli not, we measured the three IgGs with TeNT by SPR. The antigen binding affinity of 2-7G, 2-2D, and S-4-7H of the IgG was significantly higher (lower KD) than for the corresponding scFv. 2-7G, 2-2D, and S-4-7H antibodies with a KD of 5.20 × 10−11 M, 1.12 × 10−9 M and 3.57 × 10−10 M were obtained (Table 4).

View Article: PubMed Central - PubMed

ABSTRACT

Tetanus neurotoxin (TeNT) produced by Clostridiumtetani 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.

No MeSH data available.