GD2 Expression Research Articles

Variable Expression of the Disialoganglioside GD2 in Breast Cancer Molecular Subtypes.

Simple SummaryGD2 is an antigen that is tumor-specific and can be used as a target for specific immunotherapies. Since the knowledge about GD2 in breast cancer is limited, we analyzed the frequency of GD2 expression in breast cancer using two different staining methods and the impact of GD2 expression on the survival of breast cancer patients. GD2 expression was found in more than 50% of breast cancer cases, with the highest frequency in hormone receptor-positive tumors. GD2 expression was not significantly associated with patient outcome. Unlike previous studies with smaller sample sizes that lacked correlation with clinical data, this study includes a larger cohort and associations with survival data and shows that GD2 is expressed on human breast cancer cells, providing a potential target for immunotherapies (e.g., anti-GD2 antibodies or GD2 CAR T cells), that are currently undergoing clinical testing.The disialoganglioside GD2 is a tumor-associated antigen that may allow for the application of targeted immunotherapies (anti-GD2 antibodies, GD2 CAR T cells) in patients with neuroblastoma and other solid tumors. We retrospectively investigated GD2 expression in a breast cancer cohort, using immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays (TMAs), and its impact on survival. GD2 expression on IHC (n = 568) and IF (n = 503) was investigated in relation to subtypes and patient outcome. Overall, 50.2% of the 568 IHC-assessed samples and 69.8% of the 503 IF-assessed samples were GD2-positive. The highest proportion of GD2-positive tumors was observed in luminal tumors. Significantly fewer GD2-positive cases were detected in triple-negative breast cancer (TNBC) compared with other subtypes. The proportion of GD2-expressing tumors were significantly lower in HER2-positive breast cancer in comparison with luminal tumors on IF staining (but not IHC). GD2 expression of IHC or IF was not significantly associated with disease-free or overall survival, in either the overall cohort or in individual subtypes. However, GD2 expression can be seen in more than 50% of breast cancer cases, with the highest frequency in hormone receptor-positive tumors. With this high expression frequency, patients with GD2-positive advanced breast cancer of all subtypes may benefit from GD2-targeting immunotherapies, which are currently subject to clinical testing.

Multiple Immunofluorescence Imaging Analysis Reveals Differential Expression of Disialogangliosides GD3 and GD2 in Neuroblastomas.

Peripheral neuroblastic tumors (pNTs) are the most common childhood extracranial solid tumors. There are several therapeutic strategies targeting disialoganglioside GD2. Disialoganglioside GD3 has become a potential target. However, the mechanism by which pNTs express GD3 and GD2 remains unclear. We investigated the combined expression status of GD3 and GD2 in pNTs and delineated their clinicopathological values. GD3 and GD2 expression was examined in pNT tissue samples (n = 35) using immunohistochemistry and multiple immunofluorescence imaging. GD3 and GD2 expression was positive in 32/35 and 25/35 samples, respectively. Combinatorial analysis of GD3 and GD2 expression in neuroblastoma showed that both were heterogeneously expressed from cell to cell. There were higher numbers of GD3-positive and GD2-negative cells in the low-risk group than in the intermediate-risk (P = 0.014) and high-risk (P = 0.009) groups. Cases with high proportions of GD3-positive and GD2-negative cells were associated with the International Neuroblastoma Staging System stage (P = 0.004), Children's Oncology Group risk group (P = 0.001), and outcome (P = 0.019) and tended to have a higher overall survival rate. We demonstrated that neuroblastomas from low-risk patients included more GD3-positive and GD2-negative cells than those from high-risk patients. Clarifying the heterogeneity of neuroblastoma aids in better understanding the biological characteristics and clinical behavior.

Expression Analysis of GD2 by Immunohistochemistry in Invasive Breast Carcinoma: Clinical and Pathologic Correlation.

The glycosphingolipid disialoganglioside GD2 is a cell surface-associated antigen expressed on tumors of neuroectodermal origin that serves as a target of immunotherapy in select cancer types. Information about the expression of GD2 in breast cancer is limited. In the present study, we investigate the utility of GD2 as a potential biomarker for targeted treatment. The study cohort consists of 386 breast carcinomas of several histologic types. GD2 expression was assessed in both whole tumor sections and tissue microarrays with anti-GD2 3F8 monoclonal antibody immunohistochemistry and correlated with clinicopathologic features and survival outcomes. A total of 134 (35%) breast carcinomas were positive for GD2, with a median H-score of 100. 3F8 staining displayed granular and predominantly cytoplasmic or perinuclear patterns, which was confined to the neoplastic tissue in nearly all cases. GD2 positivity was significantly associated with tumor histologic type (P=0.0015), low grade (P<0.0001), estrogen receptor positivity (P<0.0001), low stage (P=0.0014), and multifocality (P=0.022). Event-free survival and overall survival of patients with GD2-positive and GD2-negative tumors were not significantly different. Our results support further assessment of GD2 using the 3F8 antibody as a predictive and prognostic biomarker in breast cancer.

CD47-SIRPα Checkpoint Inhibition Enhances Neutrophil-Mediated Killing of Dinutuximab-Opsonized Neuroblastoma Cells.

Simple SummaryCurrent immunotherapy for high-risk neuroblastoma patients involves treatment with anti-GD2 antibody dinutuximab, which has significantly improved the survival rate. Still, approximately half of the patients succumb to the tumor; therefore, efforts to improve their prognosis are urgently needed. Since T cell targeting immune checkpoint inhibitors in neuroblastoma are limited due to the low immunogenicity of these tumors, alternative immunotherapeutic approaches should be studied. The therapeutic targeting of the innate immune checkpoint CD47-SIRPα has the ability to enhance antitumor effects of myeloid cells, especially in the presence of cancer-opsonizing antibodies. Given that neutrophil ADCC is a dominant effector mechanism leading to the eradication of dinutuximab-opsonized neuroblastoma cells, we have investigated the therapeutic potential of anti-GD2 antibody in combination with CD47-SIRPα inhibition. We demonstrate here that the capacity of neutrophils to kill dinutuximab-opsonized neuroblastoma cells is controlled by the CD47-SIRPα axis and its disruption promotes their cytotoxic potential even further, significantly improving dinutuximab responsiveness.High-risk neuroblastoma, especially after recurrence, still has a very low survival rate. Immune checkpoint inhibitors targeting T cells have shown remarkable clinical efficacy in adult solid tumors, but their effects in pediatric cancers have been limited so far. On the other hand, targeting myeloid immune checkpoints, such as CD47-SIPRα, provide the opportunity to enhance antitumor effects of myeloid cells, including that of neutrophils, especially in the presence of cancer-opsonizing antibodies. Disialoganglioside (GD2)-expressing neuroblastoma cells targeted with anti-GD2 antibody dinutuximab are in part eradicated by neutrophils, as they recognize and bind the antibody targeted tumor cells through their Fc receptors. Therapeutic targeting of the innate immune checkpoint CD47-SIRPα has been shown to promote the potential of neutrophils as cytotoxic cells in different solid tumor indications using different cancer-targeting antibodies. Here, we demonstrate that the capacity of neutrophils to kill dinutuximab-opsonized neuroblastoma cells is also controlled by the CD47-SIRPα axis and can be further enhanced by antagonizing CD47-SIRPα interactions. In particular, CD47-SIRPa checkpoint inhibition enhanced neutrophil-mediated ADCC of dinutuximab-opsonized adrenergic neuroblastoma cells, whereas mesenchymal neuroblastoma cells may evade immune recognition by a reduction of GD2 expression. These findings provide a rational basis for targeting CD47-SIRPα interactions to potentiate dinutuximab responsiveness in neuroblastomas with adrenergic phenotype.

Neuroblastoma GD2 Expression and Computational Analysis of Aptamer-Based Bioaffinity Targeting.

Neuroblastoma (NB) is a neuroectodermal embryonic cancer that originates from primordial neural crest cells, and amongst pediatric cancers with high mortality rates. NB is categorized into high-, intermediate-, and low-risk cases. A significant proportion of high-risk patients who achieve remission have a minimal residual disease (MRD) that causes relapse. Whilst there exists a myriad of advanced treatment options for NB, it is still characterized by a high relapse rate, resulting in a reduced chance of survival. Disialoganglioside (GD2) is a lipo-ganglioside containing a fatty acid derivative of sphingosine that is coupled to a monosaccharide and a sialic acid. Amongst pediatric solid tumors, NB tumor cells are known to express GD2; hence, it represents a unique antigen for subclinical NB MRD detection and analysis with implications in determining a response for treatment. This article discusses NB MRD expression and analytical assays for GD2 detection and quantification as well as computational approaches for GD2 characterization based on high-throughput image processing and genomic data analysis.

Abstract 3146: Heterogeneity of the antigen-expression pattern in primary and recurrent glioblastoma patients

Abstract Introduction: Immunotherapy leads to promising results in the treatment of glioblastoma (GBM) patients. Recent clinical trials revealed a survival benefit of patients treated with monospecific CAR-T-cells. However, antigen escape mechanisms due to heterogeneous surface expression of target antigens still constitute a critical hurdle in the immunotherapeutic treatment of GBM and are a major reason for non-response or relapse. Therefore, we aim to characterize GBM's heterogeneity, the intratumoral distribution and changes of surface antigens in relapse. Methods: We performed immunohistochemical staining of the ten in vitro and in vivo most targeted antigens in CAR-T cell therapy (HER2, EGFRvIII, CD70, B7H3, Il13Rα2, NY-ESO1, GD2, CD133, CSPG4 and EphA2). We analyzed 35 patients, who underwent surgery for primary (PT) as well as recurrent GBM (RL). To address intrapersonal heterogeneity, we examined five fields of view per slide. As controls, we stained cerebral, cerebellar and brain stem slides of three healthy specimens. We quantified the percentage of low, medium and high antigen expressing cells on each slide by applying a specialized macro in ImageJ. We further weighted this expression score by multiplication with 1 (low), 2 (medium) or 3 (high expression). Finally, we calculated the median expression values of all tumors combined for each antigen. Summary: Median values of EGFRvIII (PT: 0.14; RL: 0.73), HER2 (PT: 0.67; RL: 4.00) and NY-ESO1 (PT: 0.00; RL: 0.16) expression were very low. Antigen expression of CSPG4 (PT: 4.67; RL: 2.78), Il13Rα2 (PT: 3.84; RL: 2.78), GD2 (PT: 7.50; RL: 27.03) and B7H3 (PT: 8.81; RL: 16.62) was mild. CD133 (PT: 52.16; RL: 56.13), CD70 (PT: 19.18; RL: 25.02) and EphA2 (PT: 60.65; RL: 55.46) showed moderate antigen expression. None of the evaluated antigens displayed high expression. Median values of HER2 (p=0.005, Cohen's r=0.48), EGFRvIII (p=0.042, Cohen's r=0.344), and GD2 (p=0.013, Cohen's r=0.42) were significantly increased in RL compared to PT. We found significant intrapersonal expression differences for each antigen. However, none of the healthy brain tissue specimens expressed any of the ten antigens. Conclusions: Inter- and intrapersonal heterogeneity constitutes a major obstacle for the implementation of GBM immune therapies which avoid antigen escape mechanisms. Citation Format: Vera Dufner, Moritz Meyer-Hofmann, Jonas Feldheim, Katja Maurus, Camelia Monoranu, Thomas Nerreter, Michael Hudecek, Carsten Hagemann, Ralf-Ingo Ernestus, Mario Löhr. Heterogeneity of the antigen-expression pattern in primary and recurrent glioblastoma patients [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 3146.

Abstract 3034: Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma

Abstract Anti-GD2 antibody therapy significantly improved high-risk neuroblastoma (NB) patient survival yet is associated with toxicity due to GD2 expression on pain fibers. Also, GD2 may be downregulated in NB following chemotherapy and relapse. It is paramount to identify novel tumor-associated antigens that are upregulated on the cell surface throughout therapy with minimal normal tissue expression. We utilized a novel cell surface capturing technology to define the NB cell surfaceome before and after chemotherapy and nominate potential targets for cell-mediated immunotherapy. Two NB patient derived xenografts (PDXs) and one cell line xenograft (IMR5) were treated with topotecan/cyclophosphamide in vivo. Treated and untreated tumors were disaggregated and N-glycoproteins on intact cells were oxidized, selectively labeled with biocytin-hydrazide, lysed, proteins digested, and cell surface peptides enriched using NeutrAvidin beads, followed by LC-MS analysis. We prioritized cell surface proteins that were abundant on all NB models and did not change under the selective pressure of chemotherapy. Our top hit was confirmed for cell surface expression by flow, evaluated for primary tumor and normal cell expression using gene expression datasets, and evaluated for functional relevance using CRISPR/CAS9 and immunotherapy targeting via chimeric antigen receptor (CAR). In two replicates, 292 cell surface proteins were identified as abundant and unchanged following chemotherapy. 34/292 proteins had high abundance of ≥70 a.u. across all xenografts, including NB surface proteins ALK and NCAM1. One protein identified was PTK7, a member of the WNT signaling pathway that is overexpressed in adult tumors. PTK7 is highly expressed in primary NB tumors with very low normal tissue expression. PTK7 protein is present on the cell surface in 17/17 NB cell lines and PDXs. While high PTK7 expression in primary NB is associated with poor patient prognosis, CRISPR/CAS9 knockout of PTK7 shows no functional effect on cell survival, proliferation or chemotherapy response. We developed a codon-optimized, second-generation CAR construct specific to PTK7. Our CAR-engineered T cells specifically bound to recombinant human-PTK7 Fc chimera. PTK7 CAR Jurkat T cells became activated in the presence of PTK7-expressing KELLY, IMR-5, and NB-1643 NB cells, and activation was not seen in the presence of PTK7 negative CMK leukemia cells or NB-1643 PTK7 knockout cells, confirming activation is specific to the CAR recognizing PTK7. Primary T cell PTK7 CAR studies are ongoing to assess cytotoxicity against NB. Multiple surface proteins remain stable on NB tumors after chemotherapy treatment. PTK7 is a potential cell surface antigen that could be utilized for immunotherapy approaches in NB while minimizing damage to normal tissues. PTK7 is robustly expressed in other pediatric and adult cancers, suggesting broader clinical relevance. Citation Format: Arhanti Sadanand, Victor Maximov, Jasmine Lee, Suttipong Suttapitugsakul, Jenny Shim, Chris Doering, Ronghu Wu, Robert Schnepp, Trent Spencer, Kelly Goldsmith. Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma [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 3034.

Abstract 2322: Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer

Abstract Nutrient deprivation is one of the hallmark conditions of a rapidly growing tumor. Rapid proliferation of tumor cells leads to increased consumption of metabolites such as glucose and glutamine resulting in metabolic stress. However, phenotypic and functional status of tumor cells under these conditions is not clear. We hypothesize that nutrient deprivation-induced metabolic stress results in a breast cancer stem cell-like (BCSC) phenotype in triple negative breast cancer (TNBC). We found that serum-deprived conditions result in significant upregulation of GD2+ cells compared to serum rich conditions: from 9%±2% to 26%±5% in MDA-MB-231 and 7%±2% to 29%±5% in SUM159 cells. Interestingly, this affect was compensated when the cells were supplemented with serum-rich medium. In addition, a positive correlation was observed between GD2+ cell number and tumor volume in vivo, suggesting metabolic stress in the growing tumors results in GD2+ phenotype in TNBC cells. Next, TNBC cells cultured under glucose deprivation or in the presence of glycolysis inhibitor, 2-deoxy glucose (2DG) showed a 2-fold increase in GD2+ cells compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 expression. Nutrient deprived conditions also showed a significant increase in the expression of stress markers ATF4 and SESN2 demonstrating TNBC cells under metabolic stress. In addition, the above conditions also showed a 2.5-fold upregulation of the mammosphere formation in TNBC cells compared to nutrient rich conditions. Global metabolomics profiling identified glutathione biosynthesis to be most highly upregulated in GD2+ cells. Furthermore, we found that supplementation of glutamine (precursor for glutathione biosynthesis) increases the percentage of GD2+ cells. Carbon and nitrogen tracing using 13C- and 15N-labeled glutamine by mass spectrometry showed its direct contribution to GD2 biosynthesis. Treatment with glutamine transporter (ASCT2) inhibitor V9302 reduced GD2+ cell number by 70%-80% in TNBC cell lines and dramatically inhibited mammosphere and soft agar colony formation, and migration of TNBC cell lines. Mechanistic studies revealed that V9302 inhibits glutathione pathway and induces ROS levels in TNBC cells. Additionally, V9302 treatment inhibited AKT-ERK-mTOR-4E-BP1 pathway and combination of V9302 with paclitaxel synergizes TNBC cell killing. We also identified that, V9302 treatment induced ferroptosis (iron-dependent cell death) in TNBC cells. Finally, V9302 treatment significantly inhibited tumor growth (p&amp;lt;0.0001) in TNBC-PDX model. In conclusion, metabolic stress results in GD2+ BCSC phenotype in TNBC cells and glutamine directly contributes to GD2 biosynthesis. Targeting ASCT2 could complement conventional chemotherapy in TNBC. Citation Format: Appalaraju Jaggupilli, Stanley Ly, Khoa Nguyen, Roshan Borkar, Bin Yuan, Danthasinghe Waduge Piyarathna, Yuanqing Yan, Helen Piwnica-Worms, Henry Charles Manning, Michael Andreeff, Nagireddy Putluri, Lokesh Battula. Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer [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 2322.

Lack of GD3 synthase (St8sia1) attenuates malignant properties of gliomas in genetically engineered mouse model.

High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet‐derived growth factor B‐driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma‐bearing mice and low‐grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S‐deficient gliomas. Re‐expression of GD3S restored expression of MMP9 in primary‐cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α‐MMP9 axis. This is the first report to describe the tumor‐enhancing functions of GD3S in vivo.

Chimeric Antigen Receptor 4SCAR-GD2-Modified T Cells Targeting High-Risk and Recurrent Neuroblastoma: A Phase II Multi-Center Trial in China

Background: Neuroblastoma (NB) is one of the most common childhood malignancies responsible for 15% of pediatric cancer deaths. 70% of children with NB present with high-risk features characterized by severe metastasis and poor long-term survival even if the multimodal intensive combination therapies are used. There is ongoing challenge for developing novel therapeutic strategies for NB. GD2 is a well-characterized tumor antigen in NB. Anti-GD2 antibodies and T cells genetically modified to express GD2-specific chimeric antigen receptor (GD2-CART) have demonstrated anti-cancer activity. Here we report the safety and efficacy of a new generation of GD2-CAR (4SCAR-GD2) targeting high-risk NB with a 1-year follow-up (NCI registrations: NCT02992210 and NCT02765243).Patients and Methods: Total 34 high-risk NB patients, after confirmation for GD2 expression, are enrolled from 5 hospitals in China. These patients had undergone a series of standard therapies, including surgery, chemo- and radio-therapy before CART treatment. CD3 positive cells were selected from apheresis lymphocytes and activated before lentiviral infection. The cells were transduced with a caspase 9-inducible, safety-engineered lentivector CAR containing multiple intracellular signaling domains: GD2-scFv/CD28/CD27/CD3ζ-iCasp9. Before CART infusion, patients received cyclophosphamide and fludarabine conditioning. The CART infusion dose was at average 1.1x10^6/kg. Toxicity was determined based on NCI common toxicity criteria. The International Neuroblastoma Response Criteria were used to define response after CART treatment. The total observation time for all patients is 1 year after infusion.Results: GD2 expression was scored 0 to 4 by immunohistochemical staining of tumor sections, and total 142 specimens were stained. The results showed that 9% of specimens were negative and more than 71% scored more than 2. Total 34 patients were infused with 4SCAR-GD2 T cells: 24% of them experienced no adverse reactions (per NCI-CTC v.4.0), 50% had grade 1 and 26% had grade 2 toxicity responses including fever, skin rash and peripheral nerve pain. No grade 3 or 4 toxicity was observed. CART expansion kinetics were monitored by quantitative PCR of CAR in peripheral blood. The 4SCAR-GD2 illustrated long term persistence with the highest level of copy number detected at 30% on day 180 after CART infusion. Marked tumor regression was observed in 2 patients. At 28 days after infusion, by ultrasound the mediastinal and peritoneal tumors in one patient were reduced by 94% and 99.5%, respectively. The other patient had progressive tumor located in retroperitoneum and PET-CT showed shrinking tumor two months after CART treatment; retroperitoneal SUV decreased from 2.5 to 1.5. At the one-year observation point, 38% of patients were in stable disease, 15% had partial response and 47% were in disease progression. The 1-year overall survival rate is 74%.Conclusion: This multicenter trial demonstrates that the 4SCAR-GD2 T cells are safe and effective for treating high-risk stage IV NB children. The correlations of GD2 expression in tumor, CART kinetics in blood and treatment responses could not be statistically established, suggesting a vast disease heterogeneity. Important therapeutic correlates require a large patient cohort and long-term follow-ups to elucidate. DisclosuresYu:H3 Biomedicine Inc.: Employment.

Cancer-Associated Glycosphingolipids as Tumor Markers and Targets for Cancer Immunotherapy.

Aberrant expression of glycosphingolipids is a hallmark of cancer cells and is associated with their malignant properties. Disialylated gangliosides GD2 and GD3 are considered as markers of neuroectoderm origin in tumors, whereas fucosyl-GM1 is expressed in very few normal tissues but overexpressed in a variety of cancers, especially in small cell lung carcinoma. These gangliosides are absent in most normal adult tissues, making them targets of interest in immuno-oncology. Passive and active immunotherapy strategies have been developed, and have shown promising results in clinical trials. In this review, we summarized the current knowledge on GD2, GD3, and fucosyl-GM1 expression in health and cancer, their biosynthesis pathways in the Golgi apparatus, and their biological roles. We described how their overexpression can affect intracellular signaling pathways, increasing the malignant phenotypes of cancer cells, including their metastatic potential and invasiveness. Finally, the different strategies used to target these tumor-associated gangliosides for immunotherapy were discussed, including the use and development of monoclonal antibodies, vaccines, immune system modulators, and immune effector-cell therapy, with a special focus on adoptive cellular therapy with T cells engineered to express chimeric antigen receptors.

EPCT-14. GD2 CAR T-CELLS MEDIATE CLINICAL ACTIVITY AND MANAGEABLE TOXICITY IN CHILDREN AND YOUNG ADULTS WITH H3K27M-MUTATED DIPG AND SPINAL CORD DMG

BackgroundWe previously discovered high expression of the disialoganglioside GD2 on H3K27M+ gliomas and demonstrated preclinical efficacy of intravenous (IV) GD2-targeted chimeric antigen receptor (CAR) T-cells in preclinical models of H3K27M-mutated diffuse intrinsic pontine glioma (DIPG) and diffuse midline gliomas (DMGs). We are now conducting a Phase I clinical trial (NCT04196413) of autologous GD2-targeting CAR T-cells for H3K27M+ DIPG and spinal cord DMG. Here we present the results of subjects treated at dose level 1 (DL1; 1 million GD2-CAR T-cells/kg IV). MethodsFour patients (3 DIPG, 1 spinal DMG; ages 4–25; 1M/3F) were enrolled at DL1. Three subjects with H3K27M+ DIPG received 1e6 GD2-CAR T-cells/kg IV on study. One patient with spinal DMG enrolled but became ineligible after manufacturing and was treated on an eIND at DL1. An Ommaya reservoir was placed in all subjects for therapeutic monitoring of intracranial pressure. Subjects underwent lymphodepletion with fludarabine/cyclophosphamide and remained inpatient for at least two weeks post-infusion. ResultsAll subjects developed cytokine release syndrome (Grade 1–3) manifested by fever, tachycardia and hypotension. Other toxicities included ICANS (Grade 1–2) and neurological symptoms/signs mediated by intratumoral inflammation which we have termed Tumor Inflammation-Associated Neurotoxicity (TIAN). No evidence of on-target, off-tumor toxicity was observed in any patients. No dose-limiting toxicities occurred. CAR T cells trafficked to the CNS and were detected in CSF and blood. 3/4 patients exhibited marked improvement or resolution of neurological deficits and radiographic improvement. The patient treated on an eIND exhibited >90% reduction in spinal DMG volume but progressed by month 3. Re-treatment of this subject via intracerebroventricular administration resulted in a second reduction in spinal DMG volume by ~80%. ConclusionsGD2-CAR T-cells at DL1 demonstrate a tolerable safety profile in patients with H3K27M+ DIPG/DMG with clear signs of T-cell expansion and activity including clinical responses.

131I-GD2-ch14.18 Scintigraphy to Evaluate Option for Radioimmunotherapy in Patients with Advanced Tumors.

The tumor-selective ganglioside antigene GD2 is frequently expressed on neuroblastomas and to a lesser extent on sarcomas and neuroendocrine tumors. The aim of our study was to evaluate the tumor targeting and biodistribution of 131I-labeled chimeric GD2-antibody clone 14/18 (131I-GD2-ch14.18) in patients with late-stage disease in order to identify eligibility for radioimmunotherapy. Methods: Twenty patients (neuroblastoma, n = 9; sarcoma, n = 9; pheochromocytoma, n = 1; and neuroendocrine tumor, n = 1) were involved in this study. A 21- to 131-MBq dose (1-2 MBq/kg) of 131I-GD2-ch14.18 (0.5-1.0 mg) was injected intravenously. Planar scintigraphy was performed within 1 h from injection (day 0) and on days 1, 2, 3, and 6 or 7 to analyze tumor uptake and tracer biodistribution. Serial blood samples were collected in 4 individuals. Absorbed dose to tumor lesions and organs was calculated using OLINDA software. Results: The tumor-targeting rate on a per-patient base was 65% (13/20), with 6 of 9 neuroblastomas showing uptake of 131I-GD2-ch14.18. Tumor lesions showed maximum uptake at 20-64 h after injection (effective half-life in tumors, 33-192 h). The tumor-absorbed dose varied between 0.52 and 30.2 mGy/MBq (median, 9.08 mGy/MBq; n = 13). Visual analysis showed prominent blood-pool activity up to day 2 or 3 after injection. No pronounced uptake was observed in the bone marrow compartment or in the kidneys. Bone marrow dose was calculated at 0.09-0.18 mGy/MBq (median, 0.12 mGy/MBq), whereas blood dose was 1.1-4.7 mGy/MBq. Two patients (1 neuroblastoma and 1 pheochromocytoma) with particularly high tumor uptake underwent radioimmunotherapy using 2.3 and 2.9 GBq of 131I-GD2-ch14.18, both achieving stable disease. Overall survival was 17 and 6 mo, respectively. Conclusion:131I-GD2-ch14.18 is cleared slowly from blood, not resulting in good tumor-to-background contrast until 2 d after application. With acceptable red marrow and organ dose, radioimmunotherapy is an option for patients with high tumor uptake. However, because of the variable GD2 expression, the decision should depend on pretherapeutic dosimetry.

Anti-disialoganglioside antibody internalization by neuroblastoma cells as a mechanism of immunotherapy resistance.

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines. Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines. We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity. Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro. Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.

The Central THαβ Immunity Associated Cytokine: IL-10 Has a Strong Anti-Tumor Ability Toward Established Cancer Models In Vivo and Toward Cancer Cells In Vitro.

Immunotherapy is a promising new approach for cancer treatment. In this study, I propose to use the THαβ-mediated immune response for cancer treatment. The THαβ-mediated immune response is activated by IL-10 and IL-15. Thus, I used IL-10 and-15 as therapeutic agents in the 4T1 cell line, which is a mouse cell line of breast cancer, and the NXS2 cell line, which is a mouse cell line of neuroblastoma. Cells from 4T1 and NXS2 were subcutaneously inoculated in wild type BALB/c female mice and AJ mice, respectively, and administered cytokines or an antibody treatment at various dosages. My results showed that IL-10 and IL-15 administration led to reduction in tumor volume and increase in survival. However, traditional TH1 cytokine IFN-γ administration led to increase in tumor volume and decline in survival. Antibody treatment in conjunction with IL-10 was not significantly better than IL-10, due to the expression of GD2 on immune cells. Moreover, an anti-GD2 antibody inhibited the immune cells themselves. Additionally, I found that IL-10 was directly toxic to tumor cells in vitro. Thus, I conclude that the THαβ immunological pathway is a good treatment strategy for cancer.

Surface expression of the immunotherapeutic target GD2 in osteosarcoma depends on cell confluency.

BackgroundChimeric antigen receptor (CAR) T‐cell therapy of pediatric sarcomas is challenged by the paucity of targetable cell surface antigens. A candidate target in osteosarcoma (OS) is the ganglioside GD2, but heterogeneous expression of GD2 limits its value.AimWe aimed to identify mechanisms that upregulate GD2 target expression in OS.Methods and resultsGD2 surface expression in OS cells, studied by flow cytometry, was found to vary both among and within individual OS cell lines. Pharmacological approaches, including inhibition of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) and modulation of the protein kinase C, failed to increase GD2 expression. Instead, cell confluency was found to be associated with higher GD2 expression levels both in monolayer cultures and in tumor spheroids. The sensitivity of OS cells to targeting by GD2‐specific CAR T cells was compared in an in vitro cytotoxicity assay. Higher cell confluencies enhanced the sensitivity of OS cells to GD2‐antigen specific, CAR T‐cell‐mediated in vitro cytolysis. Mechanistic studies revealed that confluency‐dependent upregulation of GD2 expression in OS cells is mediated by increased de novo biosynthesis, through a yet unknown mechanism.ConclusionExpression of GD2 in OS cell lines is highly variable and associated with increasing cell confluency in vitro. Strategies for selective upregulation of GD2 are needed to enable effective therapeutic targeting of this antigen in OS.

Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants

BackgroundHigh-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of...

Ganglioside GD3 May Suppress the Functional Activities of Benign Skin T Cells in Cutaneous T-Cell Lymphoma.

In cutaneous T-cell lymphoma (CTCL), which arises from skin-tropic memory T cells, malignant T cells and benign T cells are confined in the same skin lesions. It is thus difficult to evaluate the phenotypic characteristics and functional activities of benign T cells in CTCL. Disialoganglioside with three glycosyl groups (GD3) is increasingly expressed on the surface of solid malignant tumor cells and takes part in tumor progression and suppression of tumor immunity. However, the role of GD3 in CTCL is not well-understood. In this study, the malignant and benign T cells in CTCL skin lesions were distinguished by flow cytometry and their phenotypic characteristics were compared with those of T cells from control skin specimens. In CTCL skin lesions, the benign T cells included limited resident memory T cells (TRM), which are sessile in skin and known to exert strong antitumor function. The benign T cells showed diminished Th17 property, and the expression of GD3 was high in the malignant T cells. The expression of GD3 in the malignant T cells inversely correlated with IL-17A production from the benign CD4 T cells. GD3 from the malignant T cells was implied to be involved in suppressing the Th17 activity of the benign T cells independent of the regulation of TRM differentiation in CTCL. Revealing the role of GD3 in inhibiting the production of IL-17A in CTCL would aid the understanding of the suppressive mechanism of the antitumor activity by malignant tumor cells.

Expression of Potential Targets for Cell-Based Therapies on Melanoma Cells.

Tumor antigen-specific redirection of cytotoxic T cells (CTLs) or natural killer (NK) cells including chimeric antigen receptor (CAR-) and T cell receptor (TCR-) cell therapy is currently being evaluated in different tumor entities including melanoma. Expression of melanoma-specific antigen recognized by the respective CAR or TCR directly or presented by HLA molecules is an indispensable prerequisite for this innovative therapy. In this study, we investigated in 168 FFPE tumor specimens of patients with stage I-IV melanoma the protein expression of HER2, TRP2, ABCB5, gp100, p53, and GD2 by immunohistochemistry (IHC). These results were correlated with clinical parameters. Membrane expression of HER2 and GD2 was also investigated in ten melanoma cell lines by flow cytometry for which corresponding tumors were analyzed by IHC. Our results demonstrated that gp100 was the most frequently overexpressed protein (61%), followed by TRP2 (50%), GD2 (38%), p53 (37%), ABCB5 (17%), and HER2 (3%). TRP2 expression was higher in primary tumors compared to metastases (p = 0.005). Accordingly, TRP2 and ABCB5 expression was significantly associated with lower tumor thickness of the primary (p = 0.013 and p = 0.025). There was no association between protein expression levels and survival in advanced melanoma patients. Flow cytometric analysis revealed abundant surface expression of GD2 and HER2 in all melanoma cell lines. The discordant HER2 expression in situ and in vitro suggests a tissue culture associated induction. In summary, our data support the use of gp100 and GD2 as a potential target for developing engineered TCR- or CAR-cell therapies, respectively, against melanoma.

Anti-GD2 antibody dinutuximab inhibits triple-negative breast tumor growth by targeting GD2+ breast cancer stem-like cells

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no effective standard therapy. Breast cancer stem-like cells (BCSCs) in primary TNBCs are reported to be responsible for...