Laurence Chatel

GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins

Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy—which we call GlycoDelete—that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.

Although IL-6 Trans-Signaling Is Sufficient To Drive Local Immune Responses, Classical IL-6 Signaling Is Obligate for the Induction of T Cell-Mediated Autoimmunity

IL-6–mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6–mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.

Pan–CC Chemokine Neutralization Restricts Splenocyte Egress and Reduces Inflammation in a Model of Arthritis

Chemokines are key regulators of leukocyte trafficking and play a crucial role under homeostatic and inflammatory conditions. Because chemokines are involved in multiple pathologies, they represent an attractive class of therapeutic targets. However, because of the redundancy of this system, neutralizing a single chemokine may be insufficient to achieve therapeutic benefit. Our strategy was to use a Fc-fusion recombinant protein form of the poxvirus-derived viral CC chemokine inhibitor protein (vCCI-Fc) that has the ability to specifically bind to multiple CC chemokines and neutralize their activity. In this study, we demonstrate first that, in vivo, vCCI-Fc prevents CC chemokine-dependent migration of macrophages into inflamed tissue of carageenan-challenged mice. We next studied this effect of inhibiting CC chemokine activity in a model more relevant to human disease, collagen-induced arthritis. Mice receiving vCCI-Fc revealed a striking retention of splenocytes, including activated and IFN-γ–secreting CD4+ and CD8+ T cells, that was associated with a concomitant decrease of cells in the draining lymph nodes. These phenomena resulted in a significant decrease in the incidence of disease and a reduction in clinical score, joint inflammation, and cartilage destruction as compared with mice receiving isotype control. Taken together, these results define a role for CC chemokines in the control of disease, as interfering with their function leads to a previously unappreciated role of controlling inflammatory cell trafficking in and out of secondary lymphoid organs.

Use of a mouse model to identify a blood biomarker for IFNγ activity in pediatric secondary hemophagocytic lymphohistiocytosis.

&NA; Life‐threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon &ggr; (IFN&ggr;) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection‐driven model of sHLH in mice, we observed that the tissue‐derived levels of IFN&ggr; are actually 500‐ to 2000‐fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFN&ggr; gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFN&ggr; activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFN&ggr;‐biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFN&ggr; activity in patients with sHLH.

Anti-CD79 Antibody Induces B Cell Anergy That Protects against Autoimmunity

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell–targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell–targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell–targeted approach predicated on the induction of B cell anergy.

Neutralization of IFN-γ reverts clinical and laboratory features in a mouse model of macrophage activation syndrome

Background: The pathogenesis of macrophage activation syndrome (MAS) is not clearly understood: a large body of evidence supports the involvement of mechanisms similar to those implicated in the setting of primary hemophagocytic lymphohistiocytosis. Objective: We sought to investigate the pathogenic role of IFN‐&ggr; and the therapeutic efficacy of IFN‐&ggr; neutralization in an animal model of MAS. Methods: We used an MAS model established in mice transgenic for human IL‐6 (IL‐6TG mice) challenged with LPS (MAS mice). Levels of IFN‐&ggr; and IFN‐&ggr;–inducible chemokines were evaluated by using real‐time PCR in the liver and spleen and by means of ELISA in plasma. IFN‐&ggr; neutralization was achieved by using the anti–IFN‐&ggr; antibody XMG1.2 in vivo. Results: Mice with MAS showed a significant upregulation of the IFN‐&ggr; pathway, as demonstrated by increased mRNA levels of Ifng and higher levels of phospho–signal transducer and activator of transcription 1 in the liver and spleen and increased expression of the IFN‐&ggr;–inducible chemokines Cxcl9 and Cxcl10 in the liver and spleen, as well as in plasma. A marked increase in Il12a and Il12b expression was also found in livers and spleens of mice with MAS. In addition, mice with MAS had a significant increase in numbers of liver CD68+ macrophages. Mice with MAS treated with an anti–IFN‐&ggr; antibody showed a significant improvement in survival and body weight recovery associated with a significant amelioration of ferritin, fibrinogen, and alanine aminotransferase levels. In mice with MAS, treatment with the anti–IFN‐&ggr; antibody significantly decreased circulating levels of CXCL9, CXCL10, and downstream proinflammatory cytokines. The decrease in CXCL9 and CXCL10 levels paralleled the decrease in serum levels of proinflammatory cytokines and ferritin. Conclusion: These results provide evidence for a pathogenic role of IFN‐&ggr; in the setting of MAS.

Tumor-Directed Blockade of CD47 with Bispecific Antibodies Induces Adaptive Antitumor Immunity

CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 “antigen sink”. A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8+ T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.

Interferon gamma (IFNg) drives disease in the TLR9-mediated cytokine storm syndrome in mice

The Cytokine Storm Syndrome (CSS) is characterized by an overwhelming activation of immune cells observed in life-threatening disorders such as familial hemophagocytic lymphohistiocytosis (fHLH) and secondary (s) HLH/macrophage activation syndrome (MAS) as well as during serious infection. However, it is not known if the CSS can be attributed to a single cytokine. Increased blood levels of interferon gamma (IFNg) in HLH and sHLH/MAS patients potentially indicate a central role for this cytokine in the CSS. Using a mouse model that mimics an infection-driven CSS (i.e., CpG-ODN), our study showed that total IFNg levels originating within organs are 500 to 2,000-fold higher than those measured in peripheral blood as CSS develops. Ablation of IFNg activity in tissues led to the amelioration of the plethora of associated CSS clinical and laboratory parameters. Furthermore, the IFNg signature gene products, CXCL9 and CXCL10, correlated with disease severity in the mouse model of CSS and patients with sHLH. Thus, anti-IFNg targeted therapy should control diseases associated with the cytokine storm and we propose the use of CXCL9 or CXCL10 as a means to monitor total IFNg activity in patients.

Abstract 2482: Neutralizing CD47 in cancer cells with dual targeting kappa/lambda bodies

Neutralizing CD47, the ‘don9t eat me signal’ hijacked by different tumor types, is a novel generally applicable therapeutic strategy. Because of a distinct mechanism of action and the ability to stimulate the innate anti-tumor immunity, CD47-neutralizing agents are poised as attractive candidates for combination therapies in association with other immunotherapies. However, the development of general CD47 antagonists could be hindered by the ubiquitous and abundant expression of CD47 on virtually all healthy cells. To overcome potential pharmacological and clinical liabilities of a general CD47 antagonist, we have developed bispecific kappa/lambda bodies, which selectively target CD47 in cancer cells. These kappa/lambda bodies: (i) are full-length bispecific IgGs, (ii) bind with high affinity and neutralize the CD47-SIRP alpha interaction in cancer cells expressing a tumor-associated antigen (TAA), and (iii) mediate efficient cell killing of TAA-positive cancer cells in vitro through Fc-dependent mechanisms such as ADCP (antibody mediated cellular phagocytosis) and ADCC (antibody mediated cellular cytotoxicity). We are currently developing two molecules of this type, one targeting CD47 and CD19 (for B cell malignancies), the other targeting CD47 and mesothelin (for various mesothelin-positive solid tumors). The efficacy of the CD47/CD19 kappa/lambda body was demonstrated in vivo, using two B-cell lymphoma xenograft models in NOD/SCID mice. We also performed a pharmacokinetics study in non-human primates with the CD47/CD19 lead candidate, with the objective of assessing the potential “antigen sink” effect related to ubiquitous CD47 expression on erythrocytes, platelets and other cells. Encouragingly, the CD47/CD19 kappa/lambda body administered in a single dose to cynomolgus monkeys, at 0.5 and 10 mg/kg, showed an acceptable pharmacokinetic profile and the absence of hematological toxicities. The example of the CD47/CD19 kappa/lambda body illustrates the power of the dual-targeting approach for addressing a ubiquitous cell surface receptor such as CD47. Citation Format: Krzysztof Masternak, Lucile Broyer, Elie Dheilly, Stefano Majocchi, Valery Moine, Giovanni Magistrelli, Francois Rousseau, Ulla Ravn, Franck Gueneau, Pauline Malinge, Sebastien Calloud, Maud Charreton-Galby, Mireille Guerrier, Nessie Costes, Nicolas Bosson, Gerard Didelot, Lucie Bernard, Vanessa Buatois, Laura Cons, Laurence Chatel, Anne Papaioannou, Zoe Johnson, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer. Neutralizing CD47 in cancer cells with dual targeting kappa/lambda bodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2482. doi:10.1158/1538-7445.AM2015-2482

Interferon gamma (IFNg) production is associated to disease parameters in TLR9-induced secondary hemophagocytic lymphohistiocytosis (sHLH) in mice

IFNg is the pivotal mediator in the murine model of primary HLH. Mice given repeated injection with the TLR9 agonist, CpG-containing oligodeoxynucleotides, develop pathology resembling the human disease, sHLH. Coadministration of an anti-IL-10 Receptor (R) monoclonal antibody (mAb) with CpG induces more severe disease characterized also by hemophagocytosis (fulminant sHLH).