Stéphane Birklé

Tumor targeting of the IL‐15 superagonist RLI by an anti‐GD2 antibody strongly enhances its antitumor potency

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor‐associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)‐2‐based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL‐2 in vitro, IL‐15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL‐2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL‐15Rα‐sushi+ domain to human IL‐15. RLI showed better biological activities than IL‐15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti‐GD2‐RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti‐GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

A Monoclonal Antibody to O-Acetyl-GD2 Ganglioside and Not to GD2 Shows Potent Anti-Tumor Activity without Peripheral Nervous System Cross-Reactivity

Background Monoclonal antibodies (mAb) against GD2 ganglioside have been shown to be effective for the treatment of neuroblastoma. Beneficial actions are, however, associated with generalized pain due to the binding of anti- GD2 mAbs to peripheral nerve fibers followed by complement activation. Neuroblastoma cells that express GD2 also express its O-acetyl derivative, O-acetyl- GD2 ganglioside (OAcGD2). Hence, we investigated the distribution of OAcGD2 in human tissues using mAb 8B6 to study the cross-reactivity of mAb 8B6 with human tissues. Methodology/Principal Findings The distribution of OAcGD2 was performed in normal and malignant tissues using an immunoperoxydase technique. Anti-tumor properties of mAb 8B6 were studied in vitro and in vivo in a transplanted tumor model in mice. We found that OAcGD2 is not expressed by peripheral nerve fibers. Furthermore, we demonstrated that mAb 8B6 was very effective in the in vitro and in vivo suppression of the growth of tumor cells. Importantly, mAb 8B6 anti-tumor efficacy was comparable to that of mAb 14G2a specific to GD2. Conclusion/Significance Development of therapeutic antibodies specific to OAcGD2 may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of antibodies.

Binding Activities and Antitumor Properties of a New Mouse/ Human Chimeric Antibody Specific for GD2 Ganglioside Antigen

Purpose: We previously generated a mouse monoclonal antibody (mAb) specific for the tumor-associated GD2 ganglioside antigen. Here, we describe the development of a chimeric anti-GD2 mAb for more effective tumor immunotherapy. Experimental Design: We cloned the cDNA encoding the immunoglobulin light and heavy chains of the 60C3 anti-GD2 mAb, and constructed chimeric genes by linking the cDNA fragments of the variable regions of the murine light and heavy chains to cDNA fragments of the human κ and γ1 constant regions, respectively. Results: The resultant chimeric anti-GD2 mAb, c.60C3, showed identical binding affinity and specificity to that of its murine counterpart. Both c.60C3 and 60C3 were rapidly internalized by tumor cells at 37°C. When human serum and human natural killer cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell cytotoxicity, respectively, c.60C3 was more effective in killing GD2-expressing tumor cells. However, c.60C3 was ineffective at inducing cell death by apoptosis, although binding of 60C3 induced apoptotic death in vitro. In an in vivo, GD2-expressing, syngeneic tumor model, i.v. injection of c.60C3, but not of 60C3, significantly suppressed tumor growth in mice (P < 0.0005). Conclusion: Immune effector functions mediated by this antibody and its potentially reduced immunogenicity make chimeric c.60C3 a promising therapeutic agent against neuroectodermic tumors.

Cell cycle arrest and apoptosis induced by O-acetyl-GD2-specific monoclonal antibody 8B6 inhibits tumor growth in vitro and in vivo.

O-Acetyl-GD2 ganglioside is suitable antigen for tumor immunotherapy with specific therapeutic antibody. Here, we investigate the anti-tumor activity of O-acetyl-GD2-specific monoclonal antibody 8B6 on O-acetyl-GD2-positive tumor cells. The results indicated that mAb 8B6 induced growth inhibition of O-acetyl-GD2-expressing tumor cell lines in vitro with features of cell cycle arrest and apoptosis. Monoclonal antibody 8B6 treatment was also very effective in suppression of tumor growth in mice by reducing the proliferation index and increasing the apoptotic index. Such a study represents a useful framework to optimize immunotherapy with O-acetyl-GD2-specific antibody in combination with chemotherapeutic agents.

Anti-Gb3 Monoclonal Antibody Inhibits Angiogenesis and Tumor Development

Inhibiting the growth of tumor vasculature represents one of the relevant strategies against tumor progression. Between all the different pro-angiogenic molecular targets, plasma membrane glycosphingolipids have been under-investigated. In this present study, we explore the anti-angiogenic therapeutic advantage of a tumor immunotherapy targeting the globotriaosylceramide Gb3. In this purpose, a monoclonal antibody against Gb3, named 3E2 was developed and characterized. We first demonstrate that Gb3 is over-expressed in proliferative endothelial cells relative to quiescent cells. Then, we demonstrate that 3E2 inhibits endothelial cell proliferation in vitro by slowing endothelial cell proliferation and by increasing mitosis duration. Antibody 3E2 is further effective in inhibiting ex vivo angiogenesis in aorta ring assays. Moreover, 3E2 treatment inhibits NXS2 neuroblastoma development and liver metastases spreading in A/J mice. Immunohistology examination of the NXS2 metastases shows that only endothelial cells, but not cancer cells express Gb3. Finally, 3E2 treatment diminishes tumor vessels density, proving a specific therapeutic action of our monoclonal antibody to tumor vasculature. Our study demonstrates that Gb3 is a viable alternative target for immunotherapy and angiogenesis inhibition.

Chimeric Antibody c.8B6 to O-Acetyl-GD2 Mediates the Same Efficient Anti-Neuroblastoma Effects as Therapeutic ch14.18 Antibody to GD2 without Antibody Induced Allodynia

Background Anti-GD2 antibody is a proven therapy for GD2-postive neuroblastoma. Monoclonal antibodies against GD2, such as chimeric mAb ch14.18, have become benchmarks for neuroblastoma therapies. Pain, however, can limit immunotherapy with anti-GD2 therapeutic antibodies like ch14.18. This adverse effect is attributed to acute inflammation via complement activation on GD2-expressing nerves. Thus, new strategies are needed for the development of treatment intensification strategies to improve the outcome of these patients. Methodology/Principal Findings We established the mouse-human chimeric antibody c.8B6 specific to OAcGD2 in order to reduce potential immunogenicity in patients and to fill the need for a selective agent that can kill neuroblastoma cells without inducing adverse neurological side effects caused by anti-GD2 antibody immunotherapy. We further analyzed some of its functional properties compared with anti-GD2 ch14.18 therapeutic antibody. With the exception of allodynic activity, we found that antibody c.8B6 shares the same anti-neuroblastoma attributes as therapeutic ch14.18 anti-GD2 mAb when tested in cell-based assay and in vivo in an animal model. Conclusion/Significance The absence of OAcGD2 expression on nerve fibers and the lack of allodynic properties of c.8B6–which are believed to play a major role in mediating anti-GD2 mAb dose-limiting side effects–provide an important rationale for the clinical application of c.8B6 in patients with high-risk neuroblastoma.

Targeting and killing glioblastoma with monoclonal antibody to O -acetyl GD2 ganglioside

There are still unmet medical needs in the treatment of glioblastoma, the most common and the most aggressive glioma of all brain tumors. Here, we found that O-acetyl GD2 is expressed in surgically resected human glioblastoma tissue. In addition, we demonstrated that 8B6 monoclonal antibody specific for O-acetylat GD2 could effectively inhibit glioblastoma cell proliferation in vitro and in vivo. Taken together, these results indicate that O-acetylated GD2 represents a novel antigen for immunotherapeutic-based treatment of high-grade gliomas.

Neuroblastoma chemotherapy can be augmented by immunotargeting O-acetyl-GD2 tumor-associated ganglioside

ABSTRACT Despite recent advances in high-risk neuroblastoma therapy, the prognosis for patients remains poor. In addition, many patients suffer from complications related to available therapies that are highly detrimental to their quality of life. New treatment modalities are, thus, urgently needed to further improve the efficacy and reduce the toxicity of existing therapies. Since antibodies specific for O-acetyl GD2 ganglioside display pro-apoptotic activity against neuroblastoma cells, we hypothesized that combination of immunotherapy could enhance tumor efficacy of neuroblastoma chemotherapy. We demonstrate here that combination of anti-O-acetyl GD2 monoclonal antibody 8B6 with topotecan synergistically inhibited neuroblastoma cell proliferation, as shown by the combination index values. Mechanistically, we evidence that mAb 8B6 induced plasma cell membrane lesions, consistent with oncosis. Neuroblastoma tumour cells treated with mAb 8B6 indeed showed an increased uptake of topotecan by the tumor cells and a more profound tumor cell death evidenced by increased caspase-3 activation. We also found that the combination with topotecan plus monoclonal antibody 8B6 showed a more potent anti-tumor efficacy in vivo than either agent alone. Importantly, we used low-doses of topotecan with no noticeable side effect. Our data suggest that chemo-immunotherapy combinations may improve the clinical efficacy and safety profile of current chemotherapeutic modalities of neuroblastoma.

Inhibition of tumor angiogenesis by globotriaosylceramide immunotargeting

Current antiangiogenic immunotherapeutic strategies mainly focus on the blockade of circulating cytokines or receptors that are overexpressed by endothelial cells. We proposed globotriaosylceramide (Gb3) as a viable alternative target for antiangiogenic therapies. In this setting, we developed an anti-Gb3 antibody and validated its therapeutic efficacy in metastatic tumor models.

O-acetylated gangliosides: Structure, biosynthesis, immunogenicity, functions and their potential for cancer immunotherapy

Sialic acid O-acetylation is a developmentally regulated modification of gangliosides implicated in ontogeny and tumor progression. Their existence has been underestimated in the past because of their alkali-labile nature and their transient expression. New data indicates, however, that O-acetylated gangliosides perform important function in tumor malignancy. Best studied O-acetyl-GD3 blocks the pro-apoptotic activity of GD3 and promotes survival of cancer cells. In acute lymphoblastic leukaemia cells, O-acetyl-GD3 expression level also correlates with survival and drug resistance. The recent identification of the enigmatic O-acetyltransferase opens new experimental approaches for designing novel effective therapeutics targeting drug-resistant cancer cells in acute lymphoblastic leukaemia. In addition, O-acetylated gangliosides expressed at the tumor cell surface are accessible for specific monoclonal antibodies to inhibit cell growth, to induce apoptosis, and to inhibit tumor metastasis formation. Thus, passive immunotherapy using murine or murine/human chimeric monoclonal anti-O-acetylated ganglioside antibodies are currently being investigated. Particularly, targeting of O-acetyl-GD2 could reduce the acute toxicities currently associated with anti-GD2 therapeutic antibodies. This review summarizes the molecular mechanisms involved in the biosynthesis and the expression of O-acetylated gangliosides and presents the new experimental approaches that allow the characterization of their importance in tumor progression. The different strategies used by different teams to develop specific monoclonal antibodies against these poorly immunogenic glycolipids for therapeutic application are also discussed.