Disialoganglioside GD2 Research Articles

Ketamine Infusion as an Adjunct to Opioid Analgesia in Pediatric Patients with High-Risk Neuroblastoma Undergoing Treatment with Dinutuximab: Adverse Effects and Safety in a Non-ICU Setting.

Anti-GD2 immunotherapy has improved outcomes for children with high-risk neuroblastoma (HRNBL). Dinutuximab promotes complement-mediated reaction against disialoganglioside GD2, which is expressed in peripheral nerves and over-expressed in neuroblastoma. Dinutuximab is associated with ≥grade 3 neuropathic pain. Targeting GD2 stimulates the NMDA receptor, which makes ketamine useful in treatment of associated pain. The objective of this retrospective study is to describe the use of ketamine for pain uncontrolled by opioids, and ketamine's impact on total opioid usage for patients receiving dinutuximab. In addition, the secondary objective is to describe the toxicities of pain management with opioids versus opioid plus ketamine. A retrospective chart review of 40hRNBL patients receiving dinutuximab at Nationwide Children's Hospital, from 2010 to 2022, was conducted. Demographics, pain scores, medication records, and total daily IV morphine milligram equivalents (IVMME) with and without a ketamine adjunct were collected. Linear mixed effect regression was used to model IVMME use for pain management across dinutuximab cycles and explore the effect of ketamine. The study cohort included 187 dinutuximab hospitalizations from 40 patients. Age at diagnosis ranged from 1.2 to 11.4 years. 66/187hospitalizations included ketamine. The average daily IVMME during post-consolidation dinutuximab infusions was greater in admissions with ketamine (median 11.67 mg/day vs 6.09 mg/day; p = 0.0005). Ketamine was not significantly associated (p = 0.77) with daily IVMME when examining opioid use longitudinally over dinutuximab cycles and controlling for patient age. Fever/chills was more frequent in admissions that utilized ketamine (79% vs 63%; p = 0.0297). No other significant statistical differences in adverse effects were observed in patients' receiving opioids versus opioids plus ketamine. Findings suggest ketamine is safe in a non-ICU setting for treatment of complex pain during anti-GD2 immunotherapy. Additional prospective studies are needed to quantify the effect of reducing opioid side effects by including ketamine in pain management plans.

GD2 in Breast Cancer: A Potential Biomarker and Therapeutic Target.

Expression of disialoganglioside GD2 in normal tissues is primarily limited to the central nervous system, peripheral sensory nerve fibers, dermal melanocytes, lymphocytes, and mesenchymal stem cells. Its widespread overexpression in various cancer types allows it to be classified as a tumor-associated antigen with potential diagnostic and therapeutic implications. This article reviews the synthesis pathways of GD2 and its role in cancer cell adhesion, proliferation, and metastasis with a focus on breast cancer. GD2 appears to be overexpressed on the outer membrane of most breast cancer cells and breast cancer stem cells (BCSCs) and is closely linked to epithelial-mesenchymal transition (EMT). GD3 synthase (GD3S) is considered to be the rate-determining step in GD2 synthesis. Clinical studies indicate that GD2 expression is increased in 35-70% of breast cancer samples, with higher levels in triple-negative breast cancer (TNBC). This overexpression correlates with more aggressive tumor features and worse prognosis. Therapeutic targeting of GD2 with monoclonal antibodies (moABs) like dinutuximab and naxitamab has demonstrated anti-cancer activity in preclinical cancer models and human clinical trials against high-risk neuroblastoma reducing tumor growth and enhancing survival. GD2-specific chimeric antigen receptor (CAR) T-cell therapy and GD3S inhibition present other promising therapeutic strategies to improve clinical outcomes. Furthermore, GD2-targeted vaccines are currently being investigated in cancer therapy. This narrative review article underscores the critical role of GD2 in breast cancer pathogenesis and highlights the promising therapeutic opportunities it offers. It advocates for the initiation of clinical trials to further explore the potential of GD2-targeted treatment in combination with standard breast cancer therapies.

Current Knowledge and Perspectives of Immunotherapies for Neuroblastoma.

Neuroblastoma (NBL) cells highly express disialoganglioside GD2, which is restricted and weakly expressed in selected healthy cells, making it a desirable target of immunotherapy. Over the past two decades, application of dinutuximab, an anti-GD2 monoclonal antibody (mAb), has been one of the few new therapies to substantially improve outcomes to current levels. Given the persistent challenge of relapse and therapeutic resistance, there is an urgent need for new effective and tolerable treatment options for high-risk NBL. Recent breakthroughs in immune checkpoint inhibitor (ICI) therapeutics have not translated into high-risk NBL, like many other major pediatric solid tumors. Given the suppressed tumor microenvironment (TME), single ICIs like anti-CTLA4 and anti-PD1 have not demonstrated significant antitumor response rates. Meanwhile, emerging studies are reporting novel advancements in GD2-based therapies, targeted therapies, nanomedicines, and other immunotherapies such as adoptive transfer of natural killer (NK) cells and chimeric antigen receptors (CARs), and these hold interesting promise for the future of high-risk NBL patient care. Herein, we summarize the current state of the art in NBL therapeutic options and highlight the unique challenges posed by NBL that have limited the successful adoption of immune-modifying therapies. Through this review, we aim to direct the field's attention to opportunities that may benefit from a combination immunotherapy strategy.

GD2 targeting CAR T cells for neuroblastoma

Treatment of neuroblastoma is a significant clinical unmet need in paediatric oncology epitomised by high-risk disease in which relapse is common and outcomes for children with relapse or primary refractory disease are typically poor, with 4-year progression-free survival for relapse/refractory disease of 6 %. Immunotherapy targeting disialoganglioside GD2 using monoclonal antibodies (mAb) has become a component of standard of care treatment in neuroblastoma following published studies that have demonstrated clinical activity and survival benefit associated with this treatment. Hence a number of research groups have developed and clinically evaluated chimeric antigen receptor gene modified T cells (CAR-T cells) targeting GD2 in patients with relapsed and refractory neuroblastoma. Preclinical and clinical results using a range of receptor technologies and immune effectors have demonstrated the basic safety and feasibility of this approach, progressing into clinical data exhibiting promise for sustained patient benefit.

GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Abstract 5472: Direct cytotoxicity of hu14.18K.322A in high-risk neuroblastoma

Abstract Background: Neuroblastoma is a childhood cancer of the sympathetic nervous system and is associated with disialoganglioside GD2 overexpression. The main goal of this study is to investigate the involvement of GD2 targeting humanized antidisialoganglioside monoclonal antibody hu14.18K.322A (hu14) in high-risk neuroblastoma direct cell cytotoxicity, assess for synergy with induction chemotherapy agents, and delineate the mechanisms involved in cell death. Methods: For this study, we used a panel of GD2 expressing cell lines [SK-N-BE (2), SK-N-BE (1), LAN5, CHLA15] and a GD2 non-expressing cell line LAN6. We measured surface expression of GD2 and performed cell cycle analysis using flow cytometry. The role of hu14 in neuroblastoma cell death was assessed using cell viability assays, proliferation assays, real time PCR and western blot analysis. We measured synergy between hu14 and induction phase chemotherapy drugs. Further, we employed next generation sequencing (NGS) to analyze the transcriptomic changes in human neuroblastoma cell lines in response to hu14 treatment to understand the pathways involved and the molecular mechanisms of hu14-mediated cellular response. Results: Using a cell viability assay, we found that CHLA15 and SK-N-BE (1) cell lines were sensitive to hu14 treatment whereas SK-N-BE (2), LAN5, LAN6 were resistant. Hu14 treatment resulted in a dose dependent reduction in cell proliferation in these cell lines. We also found that hu14 treatment resulted in increased expression of apoptotic and autophagy pathway proteins, but not ferroptosis or necroptosis pathways proteins. Hu14 induced cell death was partially inhibited by a pan-caspase inhibitor, confirming that caspase-dependent pathways are involved. Western blot analysis also showed decreased levels of p53 phosphorylation in both sensitive cell lines upon hu14 treatment suggesting an important control mechanism centered on the p53 pathway. Hu14 treatment reduced the surface expression of GD2 in the sensitive cells suggesting direct inhibition of the GD2 synthesis pathway. However, PCR analysis of GD2 synthesis genes did not show any significant difference upon treatment suggesting posttranscriptional control of these genes. Furthermore, NGS analysis revealed alterations in several pathways, but these changes were distinct between the cell lines reconfirming the complexity of therapy response in high-risk Neuroblastoma. Conclusion: Taken together, our findings suggest that hu14.18K322A induces direct cell cytotoxicity in a subset of high-risk Neuroblastoma and synergizes well with standard chemotherapy drugs. Furthermore, our research highlights the complex nature of signaling pathways involved in hu14-mediated cytotoxicity that can be harnessed towards targeted therapy. Citation Format: Manu Gnanamony, Maria Thomas, Thu Hien Nguyen, Amber M. D'Souza, Pedro de Alarcon. Direct cytotoxicity of hu14.18K.322A in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5472.

Abstract 2673: Copper chelation overcomes the immunosuppressive neuroblastoma microenvironment to potentiate anti-GD2 antibody therapy

Abstract Neuroblastoma is the most frequent extra-cranial pediatric cancer with high-risk disease possessing a dismal five-year survival rate <50%. Antibody-based therapies targeting the surface disialoganglioside GD2 have shown promising anti-tumor activity in patients however efficacy is strongly hampered by immunosuppressive mechanisms present in the tumor microenvironment. Neuroblastomas typically feature low mutational burdens, limited major histocompatibility complex class I expression, increased immune checkpoint markers, sparse immune infiltration, and soluble mediators which drastically limit anti-tumor immunity. Given the emerging link between elevated copper levels and tumoral immune evasion, we explored copper chelation as a therapeutic strategy using the agents tetraethylenepentamine (TEPA) and the clinically approved analogue triethylenetetramine (TETA). Using the immunocompetent TH-MYCN model, we performed single-cell RNA sequencing supported by OPAL multiplex immunohistochemistry and cytokine profiling of resected tumors to assess cellular and molecular changes occurring after one week of TEPA treatment. Therapy was observed to successfully deplete intratumoral copper and reinvigorate anti-tumor immunity with increased infiltration and activity of pro-inflammatory immune cells, and was specifically found to induce egress and N1-polarisation of neutrophils. Further mechanistic studies in vitro revealed the sequestration of copper by neuroblastoma cells attenuated neutrophil function, which could be successfully reversed upon TEPA treatment. Findings propose a novel mechanism of immune evasion, highlighting copper chelation as a therapeutic strategy to counteract immunosuppression. Using the TH-MYCN model, TEPA was employed as a priming agent to effectively potentiate anti-GD2 antibody therapy and achieve durable tumor control. Importantly, this was associated with increased Fc-receptor-bearing natural killer and myeloid cells, which elicit tumor clearance via antibody-dependent cellular cytotoxicity. Currently indicated for the treatment of copper overload disorder Wilson’s disease, TETA was evaluated for potential repurposing as a novel immunomodulatory agent. Using the syngeneic NXS2 model, TETA exhibited an exceptional safety profile and combination therapy achieved durable tumor control with no relapses occurring after treatment cessation. This result was similarly associated with the infiltration of pro-inflammatory immune cells, including N1 neutrophils. Collectively, this study credentials copper chelation as a non-toxic strategy to disrupt the immunosuppressive tumor microenvironment and reinvigorate anti-tumour immunity. Findings provide crucial translational evidence for repurposing TETA to potentiate anti-GD2 antibody therapy and improve neuroblastoma patient responses. Citation Format: Jourdin R. Rouaen, Antonella Salerno, Fabio Luciani, Jayne Murray, Tyler Shai-Hee, Nicodemus Tedla, Michelle Haber, Toby N. Trahair, Orazio Vittorio. Copper chelation overcomes the immunosuppressive neuroblastoma microenvironment to potentiate anti-GD2 antibody therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2673.

GD2-targeting therapy: a comparative analysis of approaches and promising directions.

Disialoganglioside GD2 is a promising target for immunotherapy with expression primarily restricted to neuroectodermal and epithelial tumor cells. Although its role in the maintenance and repair of neural tissue is well-established, its functions during normal organism development remain understudied. Meanwhile, studies have shown that GD2 plays an important role in tumorigenesis. Its functions include proliferation, invasion, motility, and metastasis, and its high expression and ability to transform the tumor microenvironment may be associated with a malignant phenotype. Structurally, GD2 is a glycosphingolipid that is stably expressed on the surface of tumor cells, making it a suitable candidate for targeting by antibodies or chimeric antigen receptors. Based on mouse monoclonal antibodies, chimeric and humanized antibodies and their combinations with cytokines, toxins, drugs, radionuclides, nanoparticles as well as chimeric antigen receptor have been developed. Furthermore, vaccines and photoimmunotherapy are being used to treat GD2-positive tumors, and GD2 aptamers can be used for targeting. In the field of cell therapy, allogeneic immunocompetent cells are also being utilized to enhance GD2 therapy. Efforts are currently being made to optimize the chimeric antigen receptor by modifying its design or by transducing not only αβ T cells, but also γδ T cells, NK cells, NKT cells, and macrophages. In addition, immunotherapy can combine both diagnostic and therapeutic methods, allowing for early detection of disease and minimal residual disease. This review discusses each immunotherapy method and strategy, its advantages and disadvantages, and highlights future directions for GD2 therapy.

A novel approach to guide GD2-targeted therapy in pediatric tumors by PET and [64Cu]Cu-NOTA-ch14.18/CHO.

Background: The tumor-associated disialoganglioside GD2 is a bona fide immunotherapy target in neuroblastoma and other childhood tumors, including Ewing sarcoma and osteosarcoma. GD2-targeting antibodies proved to be effective in neuroblastoma and GD2-targeting chimeric antigen receptors (CAR)- expressing T cells as well as natural killer T cells (NKTs) are emerging. However, assessment of intra- and intertumoral heterogeneity has been complicated by ineffective immunohistochemistry as well as sampling bias in disseminated disease. Therefore, a non-invasive approach for the assessment and visualization of GD2 expression in-vivo is of upmost interest and might enable a more appropriate treatment stratification. Methods: Recently, [64Cu]Cu-NOTA-ch14.18/CHO (64Cu-GD2), a radiolabeled GD2-antibody for imaging with Positron-Emission-Tomography (PET) was developed. We here report our first clinical patients' series (n = 11) in different pediatric tumors assessed with 64Cu-GD2 PET/MRI. GD2-expression in tumors and tissue uptake in organs was evaluated by semiquantitative measurements of standardized uptake values (SUV) with PET/MRI on day 1 p.i. (n = 11) as well as on day 2 p.i. (n = 6). Results: In 8 of 9 patients with suspicious tumor lesions on PET/MRI at least one metastasis showed an increased 64Cu-GD2 uptake and a high tracer uptake (SUVmax > 10) was measured in 4 of those 8 patients. Of note, sufficient image quality with high tumor to background contrast was readily achieved on day 1. In case of 64Cu-GD2-positive lesions, an excellent tumor to background ratio (at least 6:1) was observed in bones, muscles or lungs, while lower tumor to background contrast was seen in the spleen, liver and kidneys. Furthermore, we demonstrated extensive tumor heterogeneity between patients as well as among different metastatic sites in individual patients. Dosimetry assessment revealed a whole-body dose of only 0.03 mGy/MBq (range 0.02-0.04). Conclusion: 64Cu-GD2 PET/MRI enables the non-invasive assessment of individual heterogeneity of GD2 expression, which challenges our current clinical practice of patient selection, stratification and immunotherapy application scheme for treatment with anti-GD2 directed therapies.

Co-Expression of an IL-15 Superagonist Facilitates Self-Enrichment of GD2-Targeted CAR-NK Cells and Mediates Potent Cell Killing in the Absence of IL-2.

In contrast to T lymphocytes, natural killer (NK) cells do not require prior sensitization but are rapidly activated upon encountering virally infected or neoplastic cells. In addition, NK cells can be safely applied in an allogeneic setting, making them important effector cells for the development of off-the-shelf therapeutics for adoptive cancer immunotherapy. To further enhance their therapeutic potential, here, we engineered continuously expanding NK-92 cells as a clinically relevant model to express a humanized second-generation chimeric antigen receptor (CAR) with a composite CD28-CD3ζ signaling domain (hu14.18.28.z) that targets the disialoganglioside GD2, which is expressed at high levels by neuroblastoma cells and other tumors of neuroectodermal origin. In a separate approach, we fused an IL-15 superagonist (RD-IL15) to the GD2-CAR via a P2A processing site. Lentivirally transduced NK-92/hu14.18.28.z and NK-92/hu14.18.28.z_RD-IL15 cells both displayed high and stable CAR surface expression and specific cytotoxicity toward GD2-positive tumor cells. GD2-CAR NK cells carrying the RD-IL15 construct in addition expressed the IL-15 superagonist, resulting in self-enrichment and targeted cell killing in the absence of exogenous IL-2. Furthermore, co-culture with RD-IL15-secreting GD2-CAR NK cells markedly enhanced proliferation and cytotoxicity of bystander immune cells in a paracrine manner. Our results demonstrate that GD2-CAR NK cells co-expressing the IL-15 superagonist mediate potent direct and indirect antitumor effects, suggesting this strategy as a promising approach for the further development of functionally enhanced cellular therapeutics.

IMMU-17. DEVELOPMENT OF AN INTRACRANIAL CANNULA IMPLANTATION SYSTEM FOR SERIAL LOCOREGIONAL CAR T CELL INFUSIONS IN MICE

Abstract Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies. Of particular interest is the use of chimeric antigen receptor (CAR) T cell therapy. Current clinical trials are ongoing targeting the cell surface molecules HER2, EGFR, B7-H3, IL13RA2, and the disialoganglioside GD2. While the majority of trials deliver CAR T cells into the cerebral spinal fluid with repeated doses, this has not been completed in preclinical models due to difficulty performing multiple stereotactic surgeries. To evaluate repeated locoregional delivery of CAR T cells in preclinical murine models, we established an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials. Following orthotopic injection and engraftment of tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. With this system, we tested novel CAR T cells directed against glypican 2 (GPC2), resulting in significant tumor regression in medulloblastoma model 7316-4509 (p<0.01), as well as significantly prolonged survival in thalamic diffuse midline glioma 7316-3058 (p<0.05). Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics, offering a superior preclinical model for translation to clinical trials.

IMMU-18. TARGETING GD2 IN THALAMIC DIFFUSE MIDLINE GLIOMA WITH TRANSIENT MRNA CAR T CELLS

Abstract Chimeric antigen receptor (CAR) T cells targeting the disialoganglioside GD2 have shown promise as a therapeutic for diffuse midline glioma (DMG). However, thalamic tumors have been excluded from clinical trials due to prior studies showing neurotoxicity and fatality when using virally transduced CAR T cells. We hypothesized repeated dosing of transient GD2-directed mRNA CAR T cells could be employed for safe and effective treatment of thalamic DMG. GD2-directed CAR T cells were created using mRNA and first tested against the murine thalamic DMG xenograft 7316-6349 via locoregional delivery with an indwelling catheter for repeated dosing. The previously reported fatal neurotoxicity observed in mice using lentiviral CAR T cells could be recapitulated with aggressive dosing. Four doses of 5x106 mRNA CAR T cells delivered intratumorally twice a week resulted in median overall survival of 9 days for GD2-treated mice compared to >30 days for CD19-treated controls (p<0.01). This toxicity could be avoided by decreasing the dose and timing of infusions to 2x106 mRNA CAR T cells delivered once weekly. Bioluminescent imaging showed regression of tumor in GD2-treated mice compared to CD19-treated controls (radiance fold change -3x106 versus +20x106 p/sec/cm2/sr, p<0.01). To assess survival before the onset of graft-versus-host disease, we utilized the aggressive hypermutant thalamic DMG model 7316-3058, treating with 2x106 CAR T cells intratumorally once weekly. Overall survival was significantly prolonged for 7316-3058 mice treated with GD2-directed mRNA CAR T cells compared to CD19-directed controls (median OS 48 days versus 33 days, p=0.03). Notably, non-tumor bearing mice treated with GD2-directed CAR T cells developed fatal neurotoxicity within 14 days, suggesting a very narrow therapeutic window in the brain. These data highlight the utility of titratable mRNA-based CAR T cell therapy for CNS tumors and establish GD2-directed mRNA CAR T cells as a safe and effective method for treating thalamic DMG.

Anti-tumor efficacy of anti-GD2 CAR NK-92 cells in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are rare and fatal pediatric brainstem gliomas with no cure. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have been proven effective in treating glioblastoma (GBM) in preclinical studies. However, there are no relevant studies on the CAR-NK treatment for DIPG. Our study is the first to evaluate the anti-tumor activity and safety of GD2-CAR NK-92 cells treatment for DIPG. Five patient-derived DIPG cells and primary pontine neural progenitor cell (PPC) were used to access disialoganglioside GD2 expression. Cell killing activity of GD2-CAR NK-92 cells was analyzed by in vitro cytotoxicity assays. Two DIPG patient-derived xenograft models were established to detect the anti-tumor efficacy of GD2-CAR NK-92 cells in vivo. Among the five patient-derived DIPG cells, four had high GD2 expression, and one had low GD2 expression. In in vitro assays, GD2-CAR NK-92 cells could effectively kill DIPG cells with high GD2 expression while having limited activity against DIPG cells with low GD2 expression. In in vivo assays, GD2-CAR NK-92 cells could inhibit tumor growth in TT150630 DIPG patient-derived xenograft mice (high GD2 expression) and prolong the overall survival of the mice. However, GD2-CAR NK-92 showed limited anti-tumor activity for TT190326DIPG patient-derived xenograft mice (low GD2 expression). Our study demonstrates the potential and safety of GD2-CAR NK-92 cells for adoptive immunotherapy of DIPG. The safety and anti-tumor effect of this therapy need to be further demonstrated in future clinical trials.

Specific and safe targeting of glioblastoma using switchable and logic-gated RevCAR T cells.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.

GD2-CART01 for Relapsed or Refractory High-Risk Neuroblastoma

BackgroundImmunotherapy with chimeric antigen receptor (CAR)–expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma.MethodsIn an academic, phase 1–2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01).ResultsA total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×106 CAR-positive T cells per kilogram of body weight) in the phase 1 portion of the trial, and no dose-limiting toxic effects were recorded; the recommended dose for the phase 2 portion of the trial was 10×106 CAR-positive T cells per kilogram. Cytokine release syndrome occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion (median persistence, 3 months; range, 1 to 30). Seventeen children had a response to the treatment (overall response, 63%); 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year overall survival and event-free survival were 60% and 36%, respectively.ConclusionsThe use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT03373097.)

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapydirected against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Naxitamab Activity in Neuroblastoma Cells Is Enhanced by Nanofenretinide and Nanospermidine

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is also expressed in neurons, skin melanocytes, and peripheral nerve fibers. Immunotherapy with monoclonal anti-GD2 antibodies has a proven efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. However, the strong neuro-toxicity associated with anti-GD2 antibodies administration has hindered, until now, the possibility for dose-escalation and protracted use, thus restraining their therapeutic potential. Strategies to increase the efficacy of anti-GD2 antibodies are actively sought, with the aim to enable chronic treatments that could eradicate minimal residual disease and subsequent relapses, often occurring after treatment. Here, we report that Nanofenretinide and Nanospermidine improved the expression of GD2 in neuroblastoma cells (CHP-134) and provided different effects in combination with the anti-GD2 antibody naxitamab. In particular, Nanofenretinide significantly increased the cytotoxic effect of naxitamab while Nanospermidine inhibited cell motility at extents proportional to naxitamab concentration. In neuroblastoma cells characterized by a low and heterogeneous basal expression of GD2, such as SH-SY5Y, which may represent the cell heterogeneity in tumors after chemotherapy, both Nanofenretinide and Nanospermidine increased GD2 expression in approximately 50% of cells, thus shifting the tumor population towards improved sensitivity to anti-GD2 antibodies.

First-in-Humans PET/MRI of InVivo GD2 Expression in Osteosarcoma.

Osteosarcoma is a malignant bone tumor with very limited therapeutic options ([ 1 ][1]). However, targeting the frequently overexpressed disialoganglioside GD2 was successful in preclinical studies with bispecific GD2 antibodies ([ 2 ][2]), and a clinical trial is ongoing using the clinically

Kigelia africana inhibits proliferation and induces cell death in stage 4 Neuroblastoma cell lines

Neuroblastoma (NB) is one of the most common solid pediatric tumors and especially high-risk NBs still account for about 12–15% of cancer related deaths in children. Kigelia africana (KA) is a plant used in traditional African medicine which has already shown its anti-cancer potential in several in vitro and in vivo studies. The aim of this study is to evaluate the effect of KA fruit extract on stage 4 high-risk NB cells. Therefore, NB cell lines with and without MYCN amplification and non-neoplastic cells were treated with KA fruit extract at different concentrations. The effect of KA on cell viability and apoptosis rate were assessed by bioluminescence-/fluorescence-based assays. Several proteins involved in survival, tumor growth, inflammation and metastasis were detected via western blot and immunofluorescence. Secreted cytokines were detected via ELISA. Phytochemical composition of the extract was analyzed by liquid chromatography with tandem mass spectrometry (LC/MS/MS). Our group demonstrates a dose- and time-dependent selective cytotoxic effect of KA fruit extract on NB, especially in MYCN non-amplified tumor cells, by inhibiting cell proliferation and inducing cell death. Western blot and immunofluorescence results demonstrate a regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), disialoganglioside GD2 and epidermal growth factor receptor (EGFR) in KA-treated tumor cells. Our results evidence striking anti-cancer properties of KA fruit and pave the way for further surveys on the therapeutic properties and mechanisms of action in NB.

Development of a variant of dinutuximab with enhanced antitumor efficacy and reduced induction of neuropathic pain.

Dinutuximab (ch14.18) was the first approved monoclonal antibody against the tumor‐associated antigen disialoganglioside GD2. Despite its success in treating neuroblastoma (NB), it triggers a significant amount of neuropathic pain in patients, possibly through complement‐dependent cytotoxicity (CDC). We hypothesized that modifying ch14.18 using antibody engineering techniques, such as humanization, affinity maturation, and Fc engineering, may enable the development of next‐generation GD2‐specific antibodies with reduced neuropathic pain and enhanced antitumor activity. In this study we developed the H3‐16 IgG1m4 antibody from ch14.18 IgG1. H3‐16 IgG1m4 exhibited enhanced binding activity to GD2 molecules and GD2‐positive cell lines as revealed by ELISA, and its cross‐binding activity to other gangliosides was not altered. The CDC activity of H3‐16 IgG1m4 was decreased, and the antibody‐dependent cellular cytotoxicity (ADCC) activity was enhanced. The pain response after H3‐16 IgG1m4 antibody administration was also reduced, as demonstrated using the von Frey test in Sprague–Dawley (SD) rats. In summary, H3‐16 IgG1m4 may have potential as a monoclonal antibody with reduced side effects.