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

Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

Background Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth 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. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure. Results In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay. Conclusion These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.Peer reviewe

T Cells Contribute to Tumor Progression by Favoring Pro-Tumoral Properties of Intra-Tumoral Myeloid Cells in a Mouse Model for Spontaneous Melanoma

Tumors affect myelopoeisis and induce the expansion of myeloid cells with immunosuppressive activity. In the MT/ret model of spontaneous metastatic melanoma, myeloid cells are the most abundant tumor infiltrating hematopoietic population and their proportion is highest in the most aggressive cutaneous metastasis. Our data suggest that the tumor microenvironment favors polarization of myeloid cells into type 2 cells characterized by F4/80 expression, a weak capacity to secrete IL-12 and a high production of arginase. Myeloid cells from tumor and spleen of MT/ret mice inhibit T cell proliferation and IFNγ secretion. Interestingly, T cells play a role in type 2 polarization of myeloid cells. Indeed, intra-tumoral myeloid cells from MT/ret mice lacking T cells are not only less suppressive towards T cells than corresponding cells from wild-type MT/ret mice, but they also inhibit more efficiently melanoma cell proliferation. Thus, our data support the existence of a vicious circle, in which T cells may favor cancer development by establishing an environment that is likely to skew myeloid cell immunity toward a tumor promoting response that, in turn, suppresses immune effector cell functions

Isolation of non-human primates recombinant antibody fragments neutralizing toxins and antibody epitope mapping

Les anticorps recombinants représentent une approche prometteuse pour améliorer le traitement et la prophylaxie des maladies causées par les armes biologiques. De tels anticorps peuvent être isolés à partir de primates non humains, dont l'immunisation est plus facile à concevoir et à réaliser que l'immunisation d'humains. Des chimpanzés (Pan troglodytes) et des macaques (Macaca mulatta et M. fascicularis) ont été utilisés pour de tels travaux, et notre analyse de séquences a démontré que l'utilisation de chimpanzés n'apporte pas d'avantage significatif malgré leur plus grande proximité phylogénétique avec l'Homme. La suite de ce travail a donc utilisé des macaques, plus facilement accessibles en France que les chimpanzés. Dans le cadre du projet européen AntiBotABE, des banques immunes exposées àla surface de phages ont été construites à partir de macaques (M. fascicularis) immunisés puis criblées, et des scFv neutralisant simultanément les toxines botuliques (BoNT) A1 et A2 en ciblant leurs chaines lourdes, et BoNT/E3 en ciblant sa chaine légère ont été isolés. D'autre part, un anticorps neutralisant de façon croisée la toxine létale et la toxine oedémateuse de Bacillus anthracis avait été précédemment isolé. Ses épitopes ont été localisés au cours de la présente thèse par une méthode tirant partie de cette réactivité croisée. Ils correspondent à la région [229-230]-[234-236] de la sous-unité LF (Lethal Factor) et à la région [229-230]-[234-236] de la sous-unité EF (Edema Factor). Le principe de cette localisation d'épitope pourrait être ré-employé pour localiser les épitopes des scFv neutralisant les BoNT.Recombinant antibodies represent a promising approach to improve the treatment andprophylaxis of diseases caused by bioweapons. Such antibodies may be isolated from nonhumanprimates, whose immunization is much easier to conceive and realized thanimmunization of humans. Chimpanzees (Pan troglodytes) and macaques (Macaca mulattaand M. fascicularis, particularly) have been utilized for such purposes, and our sequenceanalysis has demonstrated that using chimpanzees does not bring a significant advantagedespite their closer phylogenetic proximity with humans. The rest of this thesis has thusutilized macaques, easier to access in France than chimpanzees. In the context of theEuropean AntiBotABE project, phage-displayed immune libraries have been constructed fromimmunized macaques (M. fascicularis) then screened, and scFv simultaneously neutralizingbotulinum toxins (BoNT) A1 and A2 by targeting their heavy chains, and BoNT/E3 bytargeting its light chain were isolated. On the other side, an antibody cross-neutralizing thelethal toxin and the edema toxin of Bacillus anthracis had been formerly isolated. Its epitopeshave been mapped in the course of the present thesis by a method taking advantage of itscross-reactivity. They correspond to the [229-230]-[234-236] region of LF (Lethal Factor)subunit and to the [229-230]-[234-236] region of EF (Edema Factor) subunit. The principle ofthis epitope mapping could be re-employed to map the epitopes of BoNT-neutralizing scFv

Isolement de fragments d'anticorps recombinants neutralisant des toxines à partir de primates non humains et localisation de l'épitope d'un anticorps.

Recombinant antibodies represent a promising approach to improve the treatment andprophylaxis of diseases caused by bioweapons. Such antibodies may be isolated from nonhumanprimates, whose immunization is much easier to conceive and realized thanimmunization of humans. Chimpanzees (Pan troglodytes) and macaques (Macaca mulattaand M. fascicularis, particularly) have been utilized for such purposes, and our sequenceanalysis has demonstrated that using chimpanzees does not bring a significant advantagedespite their closer phylogenetic proximity with humans. The rest of this thesis has thusutilized macaques, easier to access in France than chimpanzees. In the context of theEuropean AntiBotABE project, phage-displayed immune libraries have been constructed fromimmunized macaques (M. fascicularis) then screened, and scFv simultaneously neutralizingbotulinum toxins (BoNT) A1 and A2 by targeting their heavy chains, and BoNT/E3 bytargeting its light chain were isolated. On the other side, an antibody cross-neutralizing thelethal toxin and the edema toxin of Bacillus anthracis had been formerly isolated. Its epitopeshave been mapped in the course of the present thesis by a method taking advantage of itscross-reactivity. They correspond to the [229-230]-[234-236] region of LF (Lethal Factor)subunit and to the [229-230]-[234-236] region of EF (Edema Factor) subunit. The principle ofthis epitope mapping could be re-employed to map the epitopes of BoNT-neutralizing scFv.Les anticorps recombinants représentent une approche prometteuse pour améliorer le traitement et la prophylaxie des maladies causées par les armes biologiques. De tels anticorps peuvent être isolés à partir de primates non humains, dont l'immunisation est plus facile à concevoir et à réaliser que l'immunisation d'humains. Des chimpanzés (Pan troglodytes) et des macaques (Macaca mulatta et M. fascicularis) ont été utilisés pour de tels travaux, et notre analyse de séquences a démontré que l'utilisation de chimpanzés n'apporte pas d'avantage significatif malgré leur plus grande proximité phylogénétique avec l'Homme. La suite de ce travail a donc utilisé des macaques, plus facilement accessibles en France que les chimpanzés. Dans le cadre du projet européen AntiBotABE, des banques immunes exposées àla surface de phages ont été construites à partir de macaques (M. fascicularis) immunisés puis criblées, et des scFv neutralisant simultanément les toxines botuliques (BoNT) A1 et A2 en ciblant leurs chaines lourdes, et BoNT/E3 en ciblant sa chaine légère ont été isolés. D'autre part, un anticorps neutralisant de façon croisée la toxine létale et la toxine oedémateuse de Bacillus anthracis avait été précédemment isolé. Ses épitopes ont été localisés au cours de la présente thèse par une méthode tirant partie de cette réactivité croisée. Ils correspondent à la région [229-230]-[234-236] de la sous-unité LF (Lethal Factor) et à la région [229-230]-[234-236] de la sous-unité EF (Edema Factor). Le principe de cette localisation d'épitope pourrait être ré-employé pour localiser les épitopes des scFv neutralisant les BoNT

Isolement de fragments d'anticorps recombinants neutralisant des toxines à partir de primates non humains et localisation de l'épitope d'un anticorps.

Recombinant antibodies represent a promising approach to improve the treatment andprophylaxis of diseases caused by bioweapons. Such antibodies may be isolated from nonhumanprimates, whose immunization is much easier to conceive and realized thanimmunization of humans. Chimpanzees (Pan troglodytes) and macaques (Macaca mulattaand M. fascicularis, particularly) have been utilized for such purposes, and our sequenceanalysis has demonstrated that using chimpanzees does not bring a significant advantagedespite their closer phylogenetic proximity with humans. The rest of this thesis has thusutilized macaques, easier to access in France than chimpanzees. In the context of theEuropean AntiBotABE project, phage-displayed immune libraries have been constructed fromimmunized macaques (M. fascicularis) then screened, and scFv simultaneously neutralizingbotulinum toxins (BoNT) A1 and A2 by targeting their heavy chains, and BoNT/E3 bytargeting its light chain were isolated. On the other side, an antibody cross-neutralizing thelethal toxin and the edema toxin of Bacillus anthracis had been formerly isolated. Its epitopeshave been mapped in the course of the present thesis by a method taking advantage of itscross-reactivity. They correspond to the [229-230]-[234-236] region of LF (Lethal Factor)subunit and to the [229-230]-[234-236] region of EF (Edema Factor) subunit. The principle ofthis epitope mapping could be re-employed to map the epitopes of BoNT-neutralizing scFv.Les anticorps recombinants représentent une approche prometteuse pour améliorer le traitement et la prophylaxie des maladies causées par les armes biologiques. De tels anticorps peuvent être isolés à partir de primates non humains, dont l'immunisation est plus facile à concevoir et à réaliser que l'immunisation d'humains. Des chimpanzés (Pan troglodytes) et des macaques (Macaca mulatta et M. fascicularis) ont été utilisés pour de tels travaux, et notre analyse de séquences a démontré que l'utilisation de chimpanzés n'apporte pas d'avantage significatif malgré leur plus grande proximité phylogénétique avec l'Homme. La suite de ce travail a donc utilisé des macaques, plus facilement accessibles en France que les chimpanzés. Dans le cadre du projet européen AntiBotABE, des banques immunes exposées àla surface de phages ont été construites à partir de macaques (M. fascicularis) immunisés puis criblées, et des scFv neutralisant simultanément les toxines botuliques (BoNT) A1 et A2 en ciblant leurs chaines lourdes, et BoNT/E3 en ciblant sa chaine légère ont été isolés. D'autre part, un anticorps neutralisant de façon croisée la toxine létale et la toxine oedémateuse de Bacillus anthracis avait été précédemment isolé. Ses épitopes ont été localisés au cours de la présente thèse par une méthode tirant partie de cette réactivité croisée. Ils correspondent à la région [229-230]-[234-236] de la sous-unité LF (Lethal Factor) et à la région [229-230]-[234-236] de la sous-unité EF (Edema Factor). Le principe de cette localisation d'épitope pourrait être ré-employé pour localiser les épitopes des scFv neutralisant les BoNT

VAR2CSA binding phenotype has ancient origin and arose before Plasmodium falciparum crossed to humans: implications in placental malaria vaccine design

International audienc