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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.


Related in: MedlinePlus

Affinities and binding kinetics of scFvs and lgGs binding TeNT. Binding curve of scFvs with TeNT, obtained using both Biacore T200. The curve was globally fit to yield equilibrium dissociation constants (KD). The equilibrium dissociation constant for each antibody-antigen pair is computed as KD = koff/kon. The results are listed in Table 3 and Table 4. (A,D,E) Binding curves of S-4-7H, 2-2D and 2-7G of the scFv with TeNT; (B,C,F) Binding curves of S-4-7H, 2-2D and 2-7G of the IgG with TeNT.
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toxins-08-00266-f002: Affinities and binding kinetics of scFvs and lgGs binding TeNT. Binding curve of scFvs with TeNT, obtained using both Biacore T200. The curve was globally fit to yield equilibrium dissociation constants (KD). The equilibrium dissociation constant for each antibody-antigen pair is computed as KD = koff/kon. The results are listed in Table 3 and Table 4. (A,D,E) Binding curves of S-4-7H, 2-2D and 2-7G of the scFv with TeNT; (B,C,F) Binding curves of S-4-7H, 2-2D and 2-7G of the IgG with TeNT.

Mentions: The affinities of 2-7G, 2-2D, and S-4-7H bound TeNT were determined by surface plasmon resonance (SPR) spectroscopy (Figure 2). 2-7G, 2-2D, and S-4-7H antibodies with a KD of 1.50 × 10−10 M, 7.82 × 10−9 M and 2.25 × 10−9 M were obtained (Table 2). The three antibody clones can easily be associated with the antigen and are difficult to dissociate. The sequencing results demonstrated that the VL genes of 2-7G and 2-2D belonged to the Vκ3 gene family, and the VH genes of 2-7G and 2-2D belonged to VH3 gene family. The VL genes of S-4-7F belonged to the VL1 gene family, and its VH genes belonged to VH6 (Table 3). Families of heavy and light chain and the CDR3-Sequences of the eight selected clones were showed as supplemental data (Tables S1 and S2).


Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library
Affinities and binding kinetics of scFvs and lgGs binding TeNT. Binding curve of scFvs with TeNT, obtained using both Biacore T200. The curve was globally fit to yield equilibrium dissociation constants (KD). The equilibrium dissociation constant for each antibody-antigen pair is computed as KD = koff/kon. The results are listed in Table 3 and Table 4. (A,D,E) Binding curves of S-4-7H, 2-2D and 2-7G of the scFv with TeNT; (B,C,F) Binding curves of S-4-7H, 2-2D and 2-7G of the IgG with TeNT.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00266-f002: Affinities and binding kinetics of scFvs and lgGs binding TeNT. Binding curve of scFvs with TeNT, obtained using both Biacore T200. The curve was globally fit to yield equilibrium dissociation constants (KD). The equilibrium dissociation constant for each antibody-antigen pair is computed as KD = koff/kon. The results are listed in Table 3 and Table 4. (A,D,E) Binding curves of S-4-7H, 2-2D and 2-7G of the scFv with TeNT; (B,C,F) Binding curves of S-4-7H, 2-2D and 2-7G of the IgG with TeNT.
Mentions: The affinities of 2-7G, 2-2D, and S-4-7H bound TeNT were determined by surface plasmon resonance (SPR) spectroscopy (Figure 2). 2-7G, 2-2D, and S-4-7H antibodies with a KD of 1.50 × 10−10 M, 7.82 × 10−9 M and 2.25 × 10−9 M were obtained (Table 2). The three antibody clones can easily be associated with the antigen and are difficult to dissociate. The sequencing results demonstrated that the VL genes of 2-7G and 2-2D belonged to the Vκ3 gene family, and the VH genes of 2-7G and 2-2D belonged to VH3 gene family. The VL genes of S-4-7F belonged to the VL1 gene family, and its VH genes belonged to VH6 (Table 3). Families of heavy and light chain and the CDR3-Sequences of the eight selected clones were showed as supplemental data (Tables S1 and S2).

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.


Related in: MedlinePlus