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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

Anti-TeNT-Hc antibodies recognize overlapping and nonoverlapping epitopes. SPR co-injection experiments were used to determine whether pairs of antibodies could bind TeNT-Hc simultaneously. The sensorgrams of all of the possible paired combinations of 2-7G, 2-2D and S-4-7H in both orientations, are shown. Dashed lines represent injection of a single antibody followed by injection of buffer. Solid lines represent co-injections of the first antibody followed by injection of a second antibody. For all experiments, 80 μL of each antibody at a concentration 20× its KD value was injected over 10,098 RUs of immobilized TeNT-Hc at 40 μL/min. In general, 2-2D and S-4-7H appeared to partial inhibition in binding according to the equation above mentioned upon injection of the second species. Conversely, 2-7G appeared to bind a distinct, nonoverlapping epitope. When 2-7G injections were followed by 2-2D and S-4-7H injections, there was an approximate doubling of total signal with the second injection. However, this was not observed with the reverse injection combinations, because the relatively fast off-rates of 2-2D and S-4-7H compared with 2-7G resulted in very significant dissociation of these TeNT-Hc from the surface before equilibrium binding of 2-7G was reached. (A) SPR co-injection experiments of 2-7G followed by 2-2D; (B) SPR co-injection experiments of 2-2D followed by 2-7G; (C) SPR co-injection experiments of 2-7G followed by S-4-7H; (D) SPR co-injection experiments of S-4-7H followed by 2-7G; (E) SPR co-injection experiments of S-4-7H followed by 2-2D; (F) SPR co-injection experiments of 2-2D followed by S-4-7H.
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toxins-08-00266-f006: Anti-TeNT-Hc antibodies recognize overlapping and nonoverlapping epitopes. SPR co-injection experiments were used to determine whether pairs of antibodies could bind TeNT-Hc simultaneously. The sensorgrams of all of the possible paired combinations of 2-7G, 2-2D and S-4-7H in both orientations, are shown. Dashed lines represent injection of a single antibody followed by injection of buffer. Solid lines represent co-injections of the first antibody followed by injection of a second antibody. For all experiments, 80 μL of each antibody at a concentration 20× its KD value was injected over 10,098 RUs of immobilized TeNT-Hc at 40 μL/min. In general, 2-2D and S-4-7H appeared to partial inhibition in binding according to the equation above mentioned upon injection of the second species. Conversely, 2-7G appeared to bind a distinct, nonoverlapping epitope. When 2-7G injections were followed by 2-2D and S-4-7H injections, there was an approximate doubling of total signal with the second injection. However, this was not observed with the reverse injection combinations, because the relatively fast off-rates of 2-2D and S-4-7H compared with 2-7G resulted in very significant dissociation of these TeNT-Hc from the surface before equilibrium binding of 2-7G was reached. (A) SPR co-injection experiments of 2-7G followed by 2-2D; (B) SPR co-injection experiments of 2-2D followed by 2-7G; (C) SPR co-injection experiments of 2-7G followed by S-4-7H; (D) SPR co-injection experiments of S-4-7H followed by 2-7G; (E) SPR co-injection experiments of S-4-7H followed by 2-2D; (F) SPR co-injection experiments of 2-2D followed by S-4-7H.

Mentions: Interestingly, 2-2D and S-4-7H antibodies both blocks binding of GD3 to the sialic acid binding site around R1226 in TeNT Hc and they could not be inhibited by each other according to a competitive binding ELISA. As we know, the advantages of a biosensor system include its ability to study molecular interactions in real time, with soluble proteins in their native state, unpurified and non-labelled. Biosensor technology instead of ELISA was chosen to determine whether 2-7G, 2-2D and S-4-7H compete in their binding to TeNT-Hc. We performed co-injection Biacore experiments with pairs of mAb (lgG), in both orientations, to determine whether antibodies could bind TeNT-Hc simultaneously (Figure 6). Of the paired combinations, only those involving 2-7G showed a significant increase in response consistent with theoretical Rmax values (~170–200 RUs) upon co-injection (Figure 6A,B). This suggests that 2-7G is free to bind TeNT-Hc when 2-2D or S-4-7H is bound and also indicates the 2-7G epitope is distinct and does not hinder binding of the other two mAbs. For 2-2D and S-4-7H only few changes in response were seen upon co-injection with theoretical Rmax values not reached, an indication that the mAbs were binding partly overlapping epitopes and hindering the binding of each other to TeNT-Hc (Figure 6C). Taken together, our Biacore based epitope mapping studies suggest 2-7G freely binds TeNT-Hc at a site that does not overlap with, or is sterically hindered by 2-2D or S-4-7H binding. 2-2D and S-4-7H bind at sites on TeNT-Hc that hinder freely accessible binding of the others, suggesting these two antibodies share bind epitopes in such close proximity to one another that it prevents unhindered interaction with sensor chip-immobilized TeNT-Hc.


Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library
Anti-TeNT-Hc antibodies recognize overlapping and nonoverlapping epitopes. SPR co-injection experiments were used to determine whether pairs of antibodies could bind TeNT-Hc simultaneously. The sensorgrams of all of the possible paired combinations of 2-7G, 2-2D and S-4-7H in both orientations, are shown. Dashed lines represent injection of a single antibody followed by injection of buffer. Solid lines represent co-injections of the first antibody followed by injection of a second antibody. For all experiments, 80 μL of each antibody at a concentration 20× its KD value was injected over 10,098 RUs of immobilized TeNT-Hc at 40 μL/min. In general, 2-2D and S-4-7H appeared to partial inhibition in binding according to the equation above mentioned upon injection of the second species. Conversely, 2-7G appeared to bind a distinct, nonoverlapping epitope. When 2-7G injections were followed by 2-2D and S-4-7H injections, there was an approximate doubling of total signal with the second injection. However, this was not observed with the reverse injection combinations, because the relatively fast off-rates of 2-2D and S-4-7H compared with 2-7G resulted in very significant dissociation of these TeNT-Hc from the surface before equilibrium binding of 2-7G was reached. (A) SPR co-injection experiments of 2-7G followed by 2-2D; (B) SPR co-injection experiments of 2-2D followed by 2-7G; (C) SPR co-injection experiments of 2-7G followed by S-4-7H; (D) SPR co-injection experiments of S-4-7H followed by 2-7G; (E) SPR co-injection experiments of S-4-7H followed by 2-2D; (F) SPR co-injection experiments of 2-2D followed by S-4-7H.
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toxins-08-00266-f006: Anti-TeNT-Hc antibodies recognize overlapping and nonoverlapping epitopes. SPR co-injection experiments were used to determine whether pairs of antibodies could bind TeNT-Hc simultaneously. The sensorgrams of all of the possible paired combinations of 2-7G, 2-2D and S-4-7H in both orientations, are shown. Dashed lines represent injection of a single antibody followed by injection of buffer. Solid lines represent co-injections of the first antibody followed by injection of a second antibody. For all experiments, 80 μL of each antibody at a concentration 20× its KD value was injected over 10,098 RUs of immobilized TeNT-Hc at 40 μL/min. In general, 2-2D and S-4-7H appeared to partial inhibition in binding according to the equation above mentioned upon injection of the second species. Conversely, 2-7G appeared to bind a distinct, nonoverlapping epitope. When 2-7G injections were followed by 2-2D and S-4-7H injections, there was an approximate doubling of total signal with the second injection. However, this was not observed with the reverse injection combinations, because the relatively fast off-rates of 2-2D and S-4-7H compared with 2-7G resulted in very significant dissociation of these TeNT-Hc from the surface before equilibrium binding of 2-7G was reached. (A) SPR co-injection experiments of 2-7G followed by 2-2D; (B) SPR co-injection experiments of 2-2D followed by 2-7G; (C) SPR co-injection experiments of 2-7G followed by S-4-7H; (D) SPR co-injection experiments of S-4-7H followed by 2-7G; (E) SPR co-injection experiments of S-4-7H followed by 2-2D; (F) SPR co-injection experiments of 2-2D followed by S-4-7H.
Mentions: Interestingly, 2-2D and S-4-7H antibodies both blocks binding of GD3 to the sialic acid binding site around R1226 in TeNT Hc and they could not be inhibited by each other according to a competitive binding ELISA. As we know, the advantages of a biosensor system include its ability to study molecular interactions in real time, with soluble proteins in their native state, unpurified and non-labelled. Biosensor technology instead of ELISA was chosen to determine whether 2-7G, 2-2D and S-4-7H compete in their binding to TeNT-Hc. We performed co-injection Biacore experiments with pairs of mAb (lgG), in both orientations, to determine whether antibodies could bind TeNT-Hc simultaneously (Figure 6). Of the paired combinations, only those involving 2-7G showed a significant increase in response consistent with theoretical Rmax values (~170–200 RUs) upon co-injection (Figure 6A,B). This suggests that 2-7G is free to bind TeNT-Hc when 2-2D or S-4-7H is bound and also indicates the 2-7G epitope is distinct and does not hinder binding of the other two mAbs. For 2-2D and S-4-7H only few changes in response were seen upon co-injection with theoretical Rmax values not reached, an indication that the mAbs were binding partly overlapping epitopes and hindering the binding of each other to TeNT-Hc (Figure 6C). Taken together, our Biacore based epitope mapping studies suggest 2-7G freely binds TeNT-Hc at a site that does not overlap with, or is sterically hindered by 2-2D or S-4-7H binding. 2-2D and S-4-7H bind at sites on TeNT-Hc that hinder freely accessible binding of the others, suggesting these two antibodies share bind epitopes in such close proximity to one another that it prevents unhindered interaction with sensor chip-immobilized TeNT-Hc.

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