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Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

View Article: PubMed Central - PubMed

ABSTRACT

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

No MeSH data available.


ELISA assay of all humanized ELC18 variants (from hu1ELC18 to hu8ELC18) and non-humanized ELC18 (No reactivity against BSA, not shown).
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toxins-08-00257-f002: ELISA assay of all humanized ELC18 variants (from hu1ELC18 to hu8ELC18) and non-humanized ELC18 (No reactivity against BSA, not shown).

Mentions: The alignment of ELC18 and the human germline genes was done by using the IMGT/V-QUEST online tool to identify the human genes that are most similar to the variable regions of ELC18 to calculate the GI. For the humanization process of ELC18 we used a multistep approach. In the first step, we designed humanized variants of the variable domains of ELC18 by exchanging AAs in the FRs that differ from the human germline sequence with their human counterpart classified as very similar and similar AA. The resulting variable domains were called hu1VH and hu1VL. In the next step, we replaced the AA classified as dissimilar AA, resulting to the humanized variants hu2VH and hu2VL and combined each variable domain with each other including the parental VH and VL. By exchanging this AA, we were able to increase the GI value of the humanized antibodies up to 97.3% (hu8ELC18) (Table 2). To validate the quality of the humanization process, we produced eight distinct variants as scFv-Fc antibodies and the antigen binding of the eight humanized variants, and the parental ELC18 was compared and validated by ELISA using an immobilized BoNT/E light chain (Figure 2). No significant difference in the antigen binding was observed between the humanized variants and the parental ELC18 (no reactivity against BSA, data not shown). For hu8ELC18, only five of the very dissimilar AAs were retained and not replaced by the human counterparts (Figure 1). For comparison, the average GI of 500 scFvs isolated from the naive human antibody gene library, HAL7/8 was 96.8% (VH), 95.4% (VL lambda) and 94.8% (VL kappa). With the highest GI value of 97.3% of all ELC18 variants, hu8ELC18 was selected for further in vivo studies. Therefore, scFv-Fc hu8ELC18 was re-cloned and produced as germline-humanized IgG.


Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody
ELISA assay of all humanized ELC18 variants (from hu1ELC18 to hu8ELC18) and non-humanized ELC18 (No reactivity against BSA, not shown).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037483&req=5

toxins-08-00257-f002: ELISA assay of all humanized ELC18 variants (from hu1ELC18 to hu8ELC18) and non-humanized ELC18 (No reactivity against BSA, not shown).
Mentions: The alignment of ELC18 and the human germline genes was done by using the IMGT/V-QUEST online tool to identify the human genes that are most similar to the variable regions of ELC18 to calculate the GI. For the humanization process of ELC18 we used a multistep approach. In the first step, we designed humanized variants of the variable domains of ELC18 by exchanging AAs in the FRs that differ from the human germline sequence with their human counterpart classified as very similar and similar AA. The resulting variable domains were called hu1VH and hu1VL. In the next step, we replaced the AA classified as dissimilar AA, resulting to the humanized variants hu2VH and hu2VL and combined each variable domain with each other including the parental VH and VL. By exchanging this AA, we were able to increase the GI value of the humanized antibodies up to 97.3% (hu8ELC18) (Table 2). To validate the quality of the humanization process, we produced eight distinct variants as scFv-Fc antibodies and the antigen binding of the eight humanized variants, and the parental ELC18 was compared and validated by ELISA using an immobilized BoNT/E light chain (Figure 2). No significant difference in the antigen binding was observed between the humanized variants and the parental ELC18 (no reactivity against BSA, data not shown). For hu8ELC18, only five of the very dissimilar AAs were retained and not replaced by the human counterparts (Figure 1). For comparison, the average GI of 500 scFvs isolated from the naive human antibody gene library, HAL7/8 was 96.8% (VH), 95.4% (VL lambda) and 94.8% (VL kappa). With the highest GI value of 97.3% of all ELC18 variants, hu8ELC18 was selected for further in vivo studies. Therefore, scFv-Fc hu8ELC18 was re-cloned and produced as germline-humanized IgG.

View Article: PubMed Central - PubMed

ABSTRACT

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

No MeSH data available.