Impairing temozolomide resistance driven by glioma stem-like cells with adjuvant immunotherapy targeting O-acetyl GD2 ganglioside.

Stem cell chemoresistance remains challenging the efficacy of the front-line temozolomide against glioblastoma. Novel therapies are urgently needed to fight those cells in order to control tumor relapse. Here, we report that anti-O-acetyl-GD2 adjuvant immunotherapy controls glioma stem-like cell-driven chemoresistance. Using patient-derived glioblastoma cells, we found that glioma stem-like cells overexpressed O-acetyl-GD2. As a result, monoclonal antibody 8B6 immunotherapy significantly increased temozolomide genotoxicity and tumor cell death in vitro by enhancing temozolomide tumor uptake. Furthermore, the combination therapy decreased the expression of the glioma stem-like cell markers CD133 and Nestin and compromised glioma stem-like cell self-renewal capabilities. When tested in vivo, adjuvant 8B6 immunotherapy prevented the extension of the temozolomide-resistant glioma stem-like cell pool within the tumor bulk in vivo and was more effective than the single agent therapies. This is the first report demonstrating that anti-O-acetyl-GD2 monoclonal antibody 8B6 targets glioblastoma in a manner that control temozolomide-resistance driven by glioma stem-like cells. Together our results offer a proof of concept for using anti-O-acetyl GD2 reagents in glioblastoma to develop more efficient combination therapies for malignant gliomas.

Interleukin-15-armored GPC3-CAR T cells for patients with solid cancers.

Interleukin-15 (IL15) promotes the survival of T lymphocytes and enhances the antitumor properties of CAR T cells in preclinical models of solid neoplasms in which CAR T cells have limited efficacy1-4. Glypican-3 (GPC3) is expressed in a group of solid cancers5-10, and here we report the first evaluation in humans of the effects of IL15 co-expression on GPC3-CAR T cells. Cohort 1 patients (NCT02905188/NCT02932956) received GPC3-CAR T cells, which were safe but produced no objective antitumor responses and reached peak expansion at two weeks. Cohort 2 patients (NCT05103631/NCT04377932) received GPC3-CAR T cells that co-expressed IL15 (15.CAR), which mediated significantly increased cell expansion and induced a disease control rate of 66% and antitumor response rate of 33%. Infusion of 15.CAR T cells was associated with increased incidence of cytokine release syndrome, which was rapidly ameliorated by activation of the inducible caspase 9 safety switch. Compared to non-responders, tumor-infiltrating 15.CAR T cells from responders showed repression of SWI/SNF epigenetic regulators and upregulation of FOS and JUN family members as well as genes related to type I interferon signaling. Collectively, these results demonstrate that IL15 increases the expansion, intratumoral survival, and antitumor activity of GPC3-CAR T cells in patients.

Glypican-3-Specific CAR T Cells Coexpressing IL15 and IL21 Have Superior Expansion and Antitumor Activity against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death in the world, and curative systemic therapies are lacking. Chimeric antigen receptor (CAR)-expressing T cells induce robust antitumor responses in patients with hematologic malignancies but have limited efficacy in patients with solid tumors, including HCC. IL15 and IL21 promote T-cell expansion, survival, and function and can improve the antitumor properties of T cells. We explored whether transgenic expression of IL15 and/or IL21 enhanced glypican-3-CAR (GPC3-CAR) T cells' antitumor properties against HCC. We previously optimized the costimulation in GPC3-CARs and selected a second-generation GPC3-CAR incorporating a 4-1BB costimulatory endodomain (GBBz) for development. Here, we generated constructs encoding IL15, IL21, or both with GBBz (15.GBBz, 21.GBBz, and 21.15.GBBz, respectively) and examined the ability of transduced T cells to kill, produce effector cytokines, and expand in an antigen-dependent manner. We performed gene-expression and phenotypic analyses of GPC3-CAR T cells and CRISPR-Cas9 knockout of the TCF7 gene. Finally, we measured GPC3-CAR T-cell antitumor activity in murine xenograft models of GPC3+ tumors. The increased proliferation of 21.15.GBBz T cells was at least in part dependent on the upregulation and maintenance of TCF-1 (encoded by TCF7) and associated with a higher percentage of stem cell memory and central memory populations after manufacturing. T cells expressing 21.15.GBBz had superior in vitro and in vivo expansion and persistence, and the most robust antitumor activity in vivo These results provided preclinical evidence to support the clinical evaluation of 21.15.GPC3-CAR T cells in patients with HCC.

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of <1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

Targeting O-Acetyl-GD2 Ganglioside for Cancer Immunotherapy.

Target selection is a key feature in cancer immunotherapy, a promising field in cancer research. In this respect, gangliosides, a broad family of structurally related glycolipids, were suggested as potential targets for cancer immunotherapy based on their higher abundance in tumors when compared with the matched normal tissues. GD2 is the first ganglioside proven to be an effective target antigen for cancer immunotherapy with the regulatory approval of dinutuximab, a chimeric anti-GD2 therapeutic antibody. Although the therapeutic efficacy of anti-GD2 monoclonal antibodies is well documented, neuropathic pain may limit its application. O-Acetyl-GD2, the O-acetylated-derivative of GD2, has recently received attention as novel antigen to target GD2-positive cancers. The present paper examines the role of O-acetyl-GD2 in tumor biology as well as the available preclinical data of anti-O-acetyl-GD2 monoclonal antibodies. A discussion on the relevance of O-acetyl-GD2 in chimeric antigen receptor T cell therapy development is also included.

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.

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.

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.