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

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.Peer reviewe

Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

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.Peer reviewe

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

International audienceThe present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms

Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B.

Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development

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

International audienceThe 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

GI value of the humanized variants of the different humanized variants of BLC7 and B2-7.

<p>GI value of the humanized variants of the different humanized variants of BLC7 and B2-7.</p

Antigen ELISA of the humanized variants of SEM120-IIIC1 against recombinant light chain of BoNT/A1.

<p>Binding of the germline-humanized anti-BoNT/A1 antibodies (hu1-hu16SEM120-IIIC1) as scFv-Fc (each 1 μg) was tested on 100 ng recombinant BoNT/A1 light chain.</p

GI value of the humanized variants of the different humanized variants of SEM120-IIIC1 and A1HC38.

<p>GI value of the humanized variants of the different humanized variants of SEM120-IIIC1 and A1HC38.</p

Protection capacities of IgGs. <i>In vivo</i> mouse paralysis assay was performed to determine protection capacities of IgGs targeting BoNT/A (5A) or BoNT/B (5B) light and heavy chains.

<p>A) Pure BoNT/A1 (0.4 LD₅₀ per dose, 1.74 pg) was pre-mixed with either hu8SEM120-IIIC1 (light chain) or hu8A1HC38 (heavy chain) at 100 μg, 50 μg, 10 μg, 5 μg or 1 μg of antibody per dose. When tested in combination IgGs (hu8SEM120-IIIC1 and hu8A1HC38 50) were pre-mixed with the same concentration of BoNT/A1 and 50 μg, 10 μg, 2.5 μg, 1.0 μg, 0.25 μg or 0.1 μg of each antibody per dose (100 μg, 20 μg, 5.0 μg, 2.0 μg, 0.5 μg or 0.2 μg total IgG). B) Complex BoNT/B2 (0.2 LD₅₀ per dose) was pre-mixed with either hu8BLC3 (light chain) or hu8B2-7 (heavy chain) at 100 μg, 10 μg, 1 μg or 0.1 μg per dose. When tested in combination IgGs (hu8BLC3 and hu8B2-7) were pre-mixed with the same concentration of BoNT/B2 and 50 μg or 0.25 μg of each antibody per dose (100 μg or 0.5 μg total IgG). In both studies mixtures of toxin and antibody were left for 30 min at room temperature before 0.1 mL was injected subcutaneously into left inguinocrural region of female MF1 strain of mice (n = 4). Animals were scored at 48 hr post injection. Results are expressed as mean score of 4 mice ± SEM. Positive control group of mice received a single injection of either BoNT/A1 (0.4 LD₅₀ per dose) or BoNT/B2 (0.2 LD₅₀ per dose). In each study negative control comprised of one group of mice (n = 4) injected with the highest concentration of IgG mixtures (50 μg each) without toxin (data not shown).</p