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Background: Mucosal-Associated Invariant T cells (MAITs) are an abundant subset of non-conventional T-cells with potent cytotoxic capacity (up to 20% of circulating T-cells) that are naturally resident in many tissues and solid tumors. T-cell redirection is a clinically validated approach to treating haematological cancers but has limited success in solid tumors. Classical T-cell engagers (TCE) bind the epsilon chain of the TCR leading to activation of all T-cells. MAIT cells utilize a semi-invariant TCR and recognize bacterial metabolites presented in the context of the MR1 protein. Biomunex has generated bispecific antibodies that bind the MAIT semi-invariant TCR (iTCR) and the HER2 receptor tyrosine kinase expressed on tumor cells. This enables the formation of an efficient immunological synapse and exclusive redirection of MAIT cells to directly kill cancer cells. Methods: Using the Biomunex proprietary BiXAb® platform, bispecific, tetravalent antibodies were generated that target the MAIT iTCR and HER2. Specific binding to the two targets was demonstrated by ELISA, DUAL ELISA and in FACS. In general, BiXAb®-mediated MAIT-cell activation, proliferation and degranulation were followed by gating on MAIT cells within a purified CD8 cell population. Tumor cell lines expressing varying amounts of HER2 were co-cultured with MAIT cells (peripheral and tumor-resident) and the BiXAb®s in several cytotoxic assays which were evaluated by measuring Chromium release, LDH release and FACS. Results: The iTCR x HER2 BiXAb® efficiently binds both target proteins with similar affinities to the parental Mabs and can bind both simultaneously, as judged by Dual ELISA. The iTCR x HER2 BiXAb® binds the MAIT-cell TCR and can bind cancer cells over a wide range of HER2 expression. BiXAb® engagement of MAIT cells and cancer cells leads to rapid activation, proliferation and degranulation of MAIT cells. In a population of PBMCs, only MAIT cells are activated by the BiXAb®. Even at low effector to target ratios (E:T = 2:1), MAIT cells efficiently kill engaged cancer cells in a HER2-dependent manner (over 50% cytotoxicity in 18 hrs). The MAIT-directed BiXAb® does not activate other T-cell subsets and hence has significantly reduced cytokine release when compared to a classical T-cell engager. Conclusions: BiXAb® mediated MAIT cell redirection leads to efficient killing of cancer cells and is a promising new approach for the treatment of solid tumors. The iTCR x HER2 BiXAb® has no impact on the general CD8 or CD4 T-cell population which may address some of the clinical limitations of classical TCEs. Citation Format: Simon Edward Plyte, Marie Fraudeau, Jonathan Grivel, Paloma Hougron, Katja Klausz, Dorothee Winterberg, Britta von Below, Alexandre Ivagnes, Claire Germain, Sebastian Amigorena, Eugene Zhukovsky, Matthias Peipp, Pierre-Emmanuel Gerard, Olivier Lantz, Julie Prigent. MAIT engagers: An efficacious novel modality in the field of T-cell engagers for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2954.

Regulatory T cells (Tregs) contribute to immunosuppression within the tumor microenvironment and have been associated with poor outcomes in a range of cancers. Targeted depletion of tumor-infiltrating Tregs (TITRs) is an attractive therapeutic strategy with the potential to enhance antitumor immunity in patients who do not respond to current treatments. However, such approaches have been limited to date by a lack of markers that are highly specific for TITRs. CCR8 is a chemokine G protein-coupled receptor (GPCR) that has recently been shown to be specifically expressed on TITRs as compared to Tregs in the periphery or other T cells within tumors. Here, we report the development and characterization of HFB101110, a humanized monoclonal antibody against CCR8 with potent and specific antibody-dependent cellular cytotoxicity (ADCC) activity. HFB101110 specifically recognizes an epitope on the N-terminus extracellular domain of CCR8 and does not recognize the closely related chemokine receptor CCR4. HFB101110 acts through a dual mechanism of action, by both depleting CCR8+ cells via ADCC and blocking binding of the CCL1 chemokine to its receptor CCR8. Blockade by HFB101110 inhibited calcium flux and chemotaxis induced by the interaction between CCL1 and CCR8. Furthermore, HFB101110 showed potent single agent anti-tumor activity associated with depletion of intratumoral Tregs in a human CCR8 knock-in mouse model. HFB101110 mediated specific ex vivo killing of Tregs from primary patient tumor samples both in the presence, and in some cases the absence, of allogeneic NK cells. HFB101110 showed favorable pharmacokinetic properties and a favorable developability profile, and was well-tolerated in both wild-type mice and cynomolgus monkeys. HFB101110 is currently being developed as a novel immunotherapy for the treatment of solid tumors coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform. Citation Format: Roshan M. Kumar, Julie Prigent, Hombline Poullain, Ayrin Kok, Carine George, Sami Ellouze, Yun-Yueh Lu, Rebecca Silver, Clarisse Monchecourt, Ross B. Fulton, Qian Zhang, Nicola Beltraminelli, Bernhard Moser, Francisco Adrian, Liang Schweizer. Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2899.

Ricin, a highly toxic protein from Ricinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.

Monoclonal antibodies targeting immune checkpoints have demonstrated clinical success in a range of tumor types; however sustained responses are achieved in a fraction of patients due to primary or secondary resistance to treatment. Recent evidence has implicated the pleiotropic immunosuppressive modulator Galactoside-binding lectin Galectin 9 (Gal-9) as a key factor in the tumor microenvironment that renders tumors resistant to current chemo and immunotherapies. High Gal-9 expression has been reported in different types of cancer, including hematological malignancies such as AML and ALL, and multiple solid tumors. Previously, we have described the characterization of an anti-human Gal-9 antibody, HFB9-2, with sub-nanomolar affinity for human, murine and cynomolgous Gal-9. HFB9-2 blocks the interaction of Gal-9 with its receptors TIM3 and CD44, leading to inhibition of Gal-9 mediated Th1 cell apoptosis and regulatory T cell expansion. HFB9-2 demonstrated significant anti-tumor efficacy and increased survival in a WEHI-164 syngeneic model as a single agent and in combination with anti-PD1 antibody. HFB9-2 is well tolerated in cynomolgous monkeys after administered by intravenous infusion as a single dose of 10, 50, 200 mg/kg, with no adverse effects and a NOAEL of 200 mg/kg. HFB9-2 developability profile assessment does not anticipate any stability and manufacturing issues. We hypothesize that targeting Gal-9 represents a valuable strategy to reduce immunosuppression and improve clinical response in selected cancer patients, in particular AML. Gal-9 has been reported to play a dual role in AML as both a self-renewal factor for leukemic stem cells and a suppressor of anti-cancer immunity. Analysis of AML patient serum samples demonstrated that Gal-9 expression was significantly higher than in healthy controls and that dropped at complete remission. Higher levels of Gal-9 were found in French-America-British (FAB) type M4 and M5 AML samples, and the lowest levels were observed in M3. To better understand the role for Gal-9 in the development of AML, we implanted primary human AML cells from different donors in M-NSG mice (NOD-Prkdcscid Il2rgem1). We demonstrate that the engraftment of primary human AML cells in these mice resulted in high levels of human Gal-9 in their serum that were detectable 2 weeks after intravenous implantation. The serum levels of Gal-9 increased over time following the increase of hCD45+ AML cells. Further analysis to evaluate the effect of Gal-9 neutralization in AML progression after primary and secondary engraftment with HFB9-2 are currently ongoing. The data presented here provide evidence that neutralization of Gal-9 with HFB9-2 blocks key immunosuppressive mechanisms known to favor cancer progression and to limit the efficacy of current immunotherapies, and together with the data obtained in AML patient samples position Gal-9 neutralization as a promising anticancer approach. Citation Format: Germain Margall, Muriel David, Julie Prigent, Dean Lee, Wenhua Xu, Joyce Pi, Xing Cai, He Zhou, Andreas Raue, Nicola Beltraminelli, Mingjie Chen, Jia Wu, Mingfang Feng, Angelo Paci, Julia Delahousse, Véronique Saada, Stéphane de Botton, Pierre Busson, Stephanie Beq, Francisco Adrian, Liang Schweizer, Yun-Yueh Lu. HFB9-2, a novel Galectin 9 neutralizing antibody for the treatment of AML and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1884.

Class-switched memory B cells are key components of the "reactive" humoral immunity, which ensures a fast and massive secretion of high-affinity antigen-specific antibodies upon antigenic challenge. In humans, IgA class-switched (IgA+ ) memory B cells and IgA antibodies are abundant in the blood. Although circulating IgA+ memory B cells and their corresponding secreted immunoglobulins likely possess major protective and/or regulatory immune roles, little is known about their specificity and function. Here, we show that IgA+ and IgG+ memory B-cell antibodies cloned from the same healthy humans share common immunoglobulin gene features. IgA and IgG memory antibodies have comparable lack of reactivity to vaccines, common mucosa-tropic viruses and commensal bacteria. However, the IgA+ memory B-cell compartment contains fewer polyreactive clones and importantly, only rare self-reactive clones compared to IgG+ memory B cells. Self-reactivity of IgAs is acquired following B-cell affinity maturation but not antibody class switching. Together, our data suggest the existence of different regulatory mechanisms for removing autoreactive clones from the IgG+ and IgA+ memory B-cell repertoires, and/or different maturation pathways potentially reflecting the distinct nature and localization of the cognate antigens recognized by individual B-cell populations.

Although monoclonal antibodies targeting immune checkpoints have demonstrated clinical success in a range of tumor types, sustained responses are only observed in a fraction of patients due to primary or secondary resistance to treatment. Recent evidence has implicated the pleiotropic immunosuppressive modulator Galactoside-binding lectin Galectin 9 (Gal-9) as a key factor present in the tumor microenvironment that renders tumors resistant to current immunotherapies. High Gal-9 expression has been reported in different types of cancer including hematological malignancies such as AML and ALL, and multiple solid tumors. We hypothesize that targeting Gal-9 may represent a valuable strategy to overcome resistance and improve clinical response in selected cancer patients. We present a monoclonal antibody, HFB9-2, that specifically binds to human Gal-9 with sub-nanomolar affinity, recognizes recombinant Gal-9 and Gal-9 produced by human tumor cells, and is cross-reactive with mouse and monkey Gal-9 orthologs. HFB9-2 blocks the interaction of Gal-9 with its receptors TIM3 and CD44 in a dose dependent manner. These two receptors have been described to mediate Gal-9-immunosuppressive signals in effector and regulatory T cells. Treatment of human PBMCs from healthy donors with HFB9-2 prevents Gal-9-induced Th1 cell apoptosis and suppresses the expansion of regulatory T cells. A humanized variant of HFB9-2, HFB9-2hz11, was generated and further characterized for its stability and pharmacokinetic profile. HFB9-2hz11 has a favorable developability profile. It demonstrated stability for at least 14 days at 40°C, as well as for several hours at low pH, and following several freeze-thaw cycles. High plasma exposures following a single dose administration to mice were observed. Furthermore, several preclinical in vivo studies are ongoing to demonstrate the efficacy of HFB-2hz11. Gal-9 has been reported to play a dual role in AML as both a self-renewal factor for leukemic stem cells and a suppressor of anti-cancer immunity, suggesting that Gal-9 neutralization represents an attractive therapeutic approach for treatment of AML patients. To explore this hypothesis, we have initiated a predictive biomarker discovery effort, using HiFiBiO's proprietary Drug Intelligent Science (DIS™) platform, using AML primary patient cells and integrating single-cell technology to identify patient subpopulations likely to respond to HFB9-2hz11. Potential novel biomarkers for patient stratification, that could be applied to other tumor indications, will be identified for AML patients. Altogether, the data presented here provide evidence that neutralization of Gal-9 with HFB9-2hz11 blocks key immunosuppressive mechanisms known to limit the efficacy of current immunotherapies and position HFB9-2hz11 as a drug candidate for clinical exploration in AML and other indications. Citation Format: Germain Margall, Rachel Pacherie, Julie Prigent, He Zhou, Francisco Adrian, Liang Schweizer, Andreas Raue, Roshan Kumar, Nicola Beltraminelli, Stéphanie Beq. HFB9-2: A novel Gal-9 neutralizing antibody to reverse immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6532.

Summary: Mucosal immune responses to HIV-1 involve the recognition of the viral envelope glycoprotein (gp)160 by tissue-resident B cells and subsequent secretion of antibodies. To characterize the B cells “sensing” HIV-1 in the gut of infected individuals, we probed monoclonal antibodies produced from single intestinal B cells binding to recombinant gp140 trimers. A large fraction of mucosal B cell antibodies were polyreactive and showed only low affinity to HIV-1 envelope glycoproteins, particularly the gp41 moiety. A few high-affinity gp140 antibodies were isolated but lacked neutralizing, potent ADCC, and transcytosis-blocking capacities. Instead, they displayed cross-reactivity with defined self-antigens. Specifically, intestinal HIV-1 gp41 antibodies targeting the heptad repeat 2 region (HR2) cluster II cross-reacted with the p38α mitogen-activated protein kinase 14 (MAPK14). Hence, physiologic polyreactivity of intestinal B cells and molecular mimicry-based self-reactivity of HIV-1 antibodies are two independent phenomena, possibly diverting and/or impairing mucosal humoral immunity to HIV-1. : Antibodies produced in mucosa after sexual transmission of HIV-1 could affect viral propagation. Planchais et al. show that intestinal B cells from HIV-1-infected individuals that recognize the HIV-1 envelope (Env) proteins are mainly low affinity and polyreactive and that rare, high-affinity antibodies to HIV-1 Env lack potent antiviral capacities and cross-react with self-antigens. Keywords: HIV-1, antibodies, B cells, mucosa, polyreactivity, cross-reactivity, MAPK14, intestine

Summary Human high-affinity antibodies to pathogens often recognize unrelated ligands. The molecular origin and the role of this polyreactivity are largely unknown. Here, we report that HIV-1 broadly neutralizing antibodies (bNAbs) are frequently polyreactive, cross-reacting with non-HIV-1 molecules, including self-antigens. Mutating bNAb genes to increase HIV-1 binding and neutralization also results in de novo polyreactivity. Unliganded paratopes of polyreactive bNAbs with improved HIV-1 neutralization exhibit a conformational flexibility, which contributes to enhanced affinity of bNAbs to various HIV-1 envelope glycoproteins and non-HIV antigens. Binding adaptation of polyreactive bNAbs to the divergent ligands mainly involves hydrophophic interactions. Plasticity of bNAbs’ paratopes may, therefore, facilitate accommodating divergent viral variants, but it simultaneously triggers promiscuous binding to non-HIV-1 antigens. Thus, a certain level of polyreactivity can be a mark of adaptable antibodies displaying optimal pathogens’ recognition., Graphical Abstract, Highlights • Most HIV-1 bNAbs are polyreactive and often cross-react with self-antigens • Polyreactivity of bNAbs involves hydrophobic interactions • Mutations enhancing HIV-1 bNAbs’ capacities also induce de novo polyreactivity • Enhanced neutralization and polyreactivity co-emerge by antibody conformational flexibility, HIV-1 bNAbs are frequently polyreactive and bind to self-antigens. Prigent et al. show that specific mutations in bNAbs that enhance their neutralizing capacity create an intrinsic structural flexibility of the antibody paratope. This promotes the conformational adaptation that facilitates binding to HIV-1 variants and polyreactivity to topologically distinct non-HIV-1 molecules.

Adult-derived human liver stem/progenitor cells (ADHLSCs) have the potential to alleviate liver injury. However, the optimal delivery route and long-term biodistribution of ADHLSCs remain unclear. In this article, we used a triple fusion reporter system to determine the kinetic differences in the biodistribution of ADHLSCs following intrasplenic (IS) and intrahepatic (IH) administration in severe combined immunodeficiency/beige mice. ADHLSCs were transduced with a lentiviral vector expressing a triple fusion reporter comprising renilla luciferase, monomeric red fluorescent protein, and truncated HSV-1 thymidine kinase. The stability and duration of the transgenes, and the effects of transduction on the cell properties were evaluated in vitro. The acute retention and long-term engraftment in vivo were revealed by positron emission tomography and bioluminescence imaging (BLI), respectively, followed by histochemical analysis. We showed that ADHLSCs can be safely transduced with the triple fusion reporter. Radiolabeled ADHLSCs showed acute cell retention at the sites of injection. The IH group showed a confined BLI signal at the injection site, while the IS group displayed a dispersed distribution at the upper abdominal liver area, and a more intense signal. In conclusion, ADHLSCs could be monitored by BLI for up to 4 weeks with a spread out biodistribution following IS injection.

There is growing evidence that cell therapy constitutes a promising strategy for liver regenerative medicine. In the setting of hepatic cancer treatments, cell therapy could prove a useful therapeutic approach for managing the acute liver failure that occurs following extended hepatectomy. In this study, we examined the influence of delivering adult-derived human liver stem/progenitor cells (ADHLSCs) at two different early time points in an immunodeficient mouse model ( Rag2−/-IL2Rg -/-) that had undergone a 70% hepatectomy procedure. The hepatic mesenchymal cells were intrasplenically infused either immediately after surgery ( n = 26) or following a critical 3-day period ( n = 26). We evaluated the cells' capacity to engraft at day 1 and day 7 following transplantation by means of human Alu qPCR quantification, along with histological assessment of human albumin and α-smooth muscle actin. In addition, cell proliferation (anti-mouse and human Ki-67 staining) and murine liver weight were measured in order to evaluate liver regeneration. At day 1 posttransplantation, the ratio of human to mouse cells was similar in both groups, whereas 1 week posttransplantation this ratio was significantly improved ( p < 0.016) in mice receiving ADHLSC injection at day 3 posthepatectomy (1.7%), compared to those injected at the time of surgery (1%). On the basis of liver weight, mouse liver regeneration was more extensive 1 week posttransplantation in mice transplanted with ADHLSCs (+65.3%) compared to that of mice from the sham vehicle group (+42.7%). In conclusion, infusing ADHLSCs 3 days after extensive hepatectomy improves the cell engraftment and murine hepatic tissue regeneration, thereby confirming that ADHLSCs could be a promising cell source for liver cell therapy and hepatic tissue repair.

Because of their high proliferative capacity, resistance to cryopreservation, and ability to differentiate into hepatocyte-like cells, stem and progenitor cells have recently emerged as attractive cell sources for liver cell therapy, a technique used as an alternative to orthotopic liver transplantation in the treatment of various hepatic ailments ranging from metabolic disorders to end-stage liver disease. Although stem and progenitor cells have been isolated from various tissues, obtaining them from the liver could be an advantage for the treatment of hepatic disorders. However, the techniques available to isolate these stem/progenitor cells are numerous and give rise to cell populations with different morphological and functional characteristics. In addition, there is currently no established consensus on the tests that need to be performed to ensure the quality and safety of these cells when used clinically. The purpose of this review is to describe the different types of liver stem/progenitor cells currently reported in the literature, discuss their suitability and limitations in terms of clinical applications, and examine how the culture and transplantation techniques can potentially be improved to achieve a better clinical outcome.

International audience; The Gram-positive, spore-forming pathogen Bacillus anthracis is the aetiological agent of anthrax. Its main virulence factors are two toxins and an anti-phagocytic capsule. When B. anthracis is grown in laboratory culture, the highest expression of the anthrax toxin genes occurs during entry into stationary phase, suggesting that nutrient limitation is an environmental cue which induces toxin production. A common bacterial response to starvation is the so-called stringent response, in which the hyperphosphorylated guanosine nucleotide (p)ppGpp is the effector molecule. In Escherichia coli, Bacillus subtilis and other bacteria, accumulation of this molecule leads to down-regulation of stable RNA synthesis and upregulation of the expression of genes involved in survival under nutrient-poor conditions. This study focuses on the stringent response of B. anthracis. We show that in B. anthracis the relA gene is responsible for the synthesis of (p)ppGpp and the stringent down-regulation of stable RNA synthesis upon starvation for the essential amino acids isoleucine, leucine and valine. The deletion of relA did not affect the expression of the virulence gene pagA or virulence in a mouse model of infection. In contrast, spore counts upon growth and sporulation in a defined medium were approximately 10,000-fold lower for the relA deletion mutant than for the parental strain. The contribution of the stringent response to efficient sporulation of B. anthracis is notable, as this suggests that the stringent response may contribute to the persistence of B. anthracis in the natural environment.

International audience; Xenotransplantation of human cells in animal models is an essential tool for evaluation of safety and efficacy of cell-based products for therapeutic use. Sensitive and reproducible methods are needed to detect and quantify human cells engrafted into the host tissue either in the targeted organ or in undesired locations. We developed a robust quantitative polymerase chain reaction (qPCR) assay based on amplification of human AluYb8 repeats, to assess the number of human cells present in rat or mouse tissues after transplantation. Standard curves of mixed human/rodent DNA and mixed human/rodent cells have been performed to determine the limit of detection and linear range of the assay. Standard curves from DNA mixing differed significantly from standard curves from cell mixing. We show here that the AluYb8 qPCR assay is highly reproducible and is able to quantify human cells in a rodent cell matrix over a large linear range that extends from 50% to 0.01% human cells. Short-term in vivo studies showed that human cells could be quantified in mouse liver up to 7 days after intrasplenic transplantation and in rat liver 4 h after intrahepatic transplantation.

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention.

International audience; Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.