Immunotherapy Of Neuroblastoma Research Articles

Combining ERAP1 silencing and entinostat therapy to overcome resistance to cancer immunotherapy in neuroblastoma

BackgroundCheckpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy.MethodsCRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence.ResultsWe found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4+ and CD8+ T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival.ConclusionsThese findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

Abstract LB221: Novel molecular targeted tumor targeted oncolytic peptide for the treatment of therapy resistant progressive neuroblastoma

Abstract Cancer treatments, particularly for solid tumors like neuroblastoma, often entail severe side effects, resistance, and the potential for secondary malignancies. To address these challenges, we explored an innovative approach employing tumor-targeting peptides. Recently, we unveiled the tumor evolution regulatory role of retinal-degeneration protein-3 (RD3) in therapy defying progressive tumors. Here in we investigated the therapeutic potential of RD3 peptides. Utilizing neuroblastoma, the most common cancer at infancy, as the tumor model, we investigated the delivery and therapeutic potential RD3 peptide. Five peptides representing unique regions, each with a C-terminal TAT tag for fluorescence and an N-terminal amide cap were developed. Peptide homing capabilities (Cell Tracking aasay) and relative potential in modifying metastatic state including invasion (Matrigel invasion assay), migration (scratch-wound assay), clonal expansion (LDTA) and tumorosphere formation (LDTA) was investigated utilizing patient derived clinical therapy defied progressive NB cells NB-EBc1 from adrenal mass and SK-N-FI from bone marrow. Whole genome RNA sequencing was performed using novogene (Illumina) platform. All peptides screened displayed tumor cell internalization and inflicted cell death at varying degrees. Among the five peptides investigated, two peptides (RD3 Pep-1 and RD3 Pep-3) profoundly inhibited cell proliferation, migration, invasion, and tumorosphere formation. A novel molecular targeted oncolytic peptide, validated through RNA sequencing, demonstrates promising efficacy for treating therapy-resistant progressive neuroblastoma. These outcomes uniquely highlight the therapeutic potential of RD3 peptides, offering a novel therapeutic candidate to counter therapy defying progressive disease. Further, the results defined the synergistic benefit RD3 peptide deliver with the current clinical maintenance therapy (GD2 immunotherapy for neuroblastoma). Together, these results identified a molecular targeted novel oncolytic peptide that could complement current standards of maintenance therapy for therapy defying progressive disease. Funding: This work was partially or in full, funded by Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-04; National Institutes of Health, NIH-P20GM103639 and; NIH-NCI- Cancer Center Support Grant P30CA225520 and Department of Defense DoD-CA210339. Citation Format: Dinesh Babu Somasundaram, Poorvi Subramanian, Sreenidhi Mohanvelu, Sheeja Aravindan, Natarajan Aravindan. Novel molecular targeted tumor targeted oncolytic peptide for the treatment of therapy resistant progressive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB221.

ALKemy to enhance chimeric antigen receptor T cell immunotherapy for neuroblastoma

Chimeric antigen receptor (CAR) Tcell immunotherapy in solid cancer is severely limited by the absence of ideal targets. In this issue of Cancer Cell, Bergaggio etal. find that anaplastic lymphoma kinase (ALK) inhibitors can enhance the function of ALK-specific CAR Tcells against neuroblastoma by increasing target density in cancer cells.

Machine learning methods revealed the roles of immune-metabolism related genes in immune infiltration, stemness, and prognosis of neuroblastoma.

Immunometabolism plays an important role in neuroblastoma (NB). However, the mechanism of immune-and metabolism-related genes (IMRGs) in NB remains unclear. This study aimed to explore the effects of IMRGs on the prognosis, immune infiltration and stemness of patients with NB using machine learning methods. R software (v4.2.1) was used to identify the differentially expressed IMRGs, and machine learning algorithm was used to screen the prognostic genes from IMRGs. Then we constructed a prognostic model and calculated the risk scores. The NB patients were grouped according to the prognosis scores. In addition, the genes most associated with the immune infiltration and stemness of NB were analyzed by weighted gene co-expression network analysis (WGCNA). There were 89 differentially expressed IMRGs between the MYCN amplification and the MYCN non-amplification group, among which CNR1, GNAI1, GLDC and ABCC4 were selected by machine learning algorithm to construct the prognosis model due to their better prediction effect. Both the K-M survival curve and the 5-year Receiver operating characteristic (ROC) curve indicated that the prognosis model could predict the prognosis of NB patients, and there was significant difference in immune infiltration between the two groups according to the median of risk score. We verified the effects of IMRGs on the prognosis, immune infiltration and stemness of NB. These findings could provide help for predicting prognosis and developing immunotherapy in NB.

The yes-associated protein (YAP) is associated with resistance to anti-GD2 immunotherapy in neuroblastoma through downregulation of ST8SIA1

ABSTRACT Pediatric patients with high-risk neuroblastoma often relapse with chemotherapy-resistant, incurable disease. Relapsed neuroblastomas harbor chemo-resistant mesenchymal tumor cells and increased expression/activity of the transcriptional co-regulator, the Yes-Associated Protein (YAP). Patients with relapsed neuroblastoma are often treated with immunotherapy such as the anti-GD2 antibody, dinutuximab, in combination with chemotherapy. We have previously shown that YAP mediates both chemotherapy and MEK inhibitor resistance in relapsed RAS mutated neuroblastoma and so posited that YAP might also be involved in anti-GD2 antibody resistance. We now show that YAP genetic inhibition significantly enhances sensitivity of mesenchymal neuroblastomas to dinutuximab and gamma delta (γδ) T cells both in vitro and in vivo. Mechanistically, YAP inhibition induces increased GD2 cell surface expression through upregulation of ST8SIA1, the gene encoding GD3 synthase and the rate-limiting enzyme in GD2 biosynthesis. The mechanism of ST8SIA1 suppression by YAP is independent of PRRX1 expression, a mesenchymal master transcription factor, suggesting YAP may be the downstream effector of mesenchymal GD2 resistance. These results therefore identify YAP as a therapeutic target to augment GD2 immunotherapy responses in patients with neuroblastoma.

Integrative analysis of multi-omics data for discovery of ferroptosis-related gene signature predicting immune activity in neuroblastoma.

Immunotherapy for neuroblastoma remains unsatisfactory due to heterogeneity and weak immunogenicity. Exploring powerful signatures for the evaluation of immunotherapy outcomes remain the primary purpose. We constructed a ferroptosis-related gene (FRG) signature by least absolute shrinkage and selection operator and Cox regression, identified 10 independent prognostic FRGs in a training cohort (GSE62564), and then verified them in an external validation cohort (TCGA). Associated with clinical factors, the signature accurately predicts overall survival of 3, 5, and 10years. An independent prognostic nomogram, which included FRG risk, age, stage of the International Neuroblastoma Staging System, and an MYCN status, was constructed. The area under the curves showed satisfactory prognostic predicting performance. Through bulk RNA-seq and proteomics data, we revealed the relationship between hub genes and the key onco-promoter MYCN gene and then validated the results in MYCN-amplified and MYCN-non-amplified cell lines with qRT-PCR. The FRG signature significantly divided patients into high- and low-risk groups, and the differentially expressed genes between the two groups were enriched in immune actions, autophagy, and carcinogenesis behaviors. The low-risk group embodied higher positive immune component infiltration and a higher expression of immune checkpoints with a more favorable immune cytolytic activity (CYT). We verified the predictive power of this signature with data from melanoma patients undergoing immunotherapy, and the predictive power was satisfactory. Gene mutations were closely related to the signature and prognosis. AURKA and PRKAA2 were revealed to be nodal hub FRGs in the signature, and both were shown to have significantly different expressions between the INSS stage IV and other stages after immunohistochemical validation. With single-cell RNA-seq analysis, we found that genes related to T cells were enriched in TNFA signaling and interferon-γ hallmark. In conclusion, we constructed a ferroptosis-related gene signature that can predict the outcomes and work in evaluating the effects of immunotherapy.

Neuroblastoma: Emerging trends in pathogenesis, diagnosis, and therapeutic targets

Neuroblastoma (NB) accounts for about 13% of all pediatric cancer mortality and is the leading cause of pediatric cancer death for children aged 1 to 5 years. NB, a developmental malignancy of neural ganglia, originates from neural crest-derived cells, which undergo a defective sympathetic neuronal differentiation due to genomic and epigenetic aberrations. NB is a complex disease with remarkable biological and genetic variation and clinical heterogeneity, such as spontaneous regression, treatment resistance, and poor survival rates. Depending on its severity, NB is categorized as high-risk, intermediate-risk, and low-risk., whereas high-risk NB accounts for a high infant mortality rate. Several studies revealed that NB cells suppress immune cell activity through diverse signaling pathways, including exosome-based signaling pathways. Exosome signaling has been shown to modulate gene expression in the target immune cells and attenuate the signaling events through non-coding RNAs. Since high-risk NB is characterized by a low survival rate and high clinical heterogeneity with current intensive therapies, it is crucial to unravel the molecular events of pathogenesis and develop novel therapeutic targets in high-risk, relapsed, or recurrent tumors in NB to improve patient survival. This article discusses etiology, pathophysiology, risk assessment, molecular cytogenetics, and the contribution of extracellular vesicles, non-coding RNAs, and cancer stem cells in the tumorigenesis of NB. We also detail the latest developments in NB immunotherapy and nanoparticle-mediated drug delivery treatment options.

Abstract 4093: Dual targeted CAR immunotherapy for neuroblastoma using γδ T cells

Abstract Cellular immunotherapy for aggressive pediatric solid tumors like neuroblastoma (NB) has focused on autologous products of αβ T cells, that so far, have been uniformly unsuccessful. γδ T cells offer a potentially superior, off-the-shelf therapy that is directly cytotoxic towards tumors without alloreactivity. Furthermore, γδ T cell infiltration of the hostile, solid tumor microenvironment is a prognostic marker of favorable disease outcome. Our team recently opened a first-in-child evaluation of unengineered allogeneic γδ T cells in combination with dinutuximab, an anti-GD2 antibody, and chemotherapy (NCT05400603). Our focus now is to optimize a second-generation γδ T-cell therapy by engineering the expression of tumor targeting chimeric antigen receptors (CAR). We hypothesize that CAR-targeting will further enhance γδ T-cell homing and antitumor potency. While anti-GD2 antibodies have been clinically successful immunotherapies for NB, GD2-targeted cell therapies need improvement. Our team recently validated a novel immunotherapy target for NB, protein tyrosine kinase 7 (PTK7), an inactive tyrosine kinase expressed highly amongst all NBs with low to no normal pediatric tissue expression. We designed a dual-targeting platform directed against both GD2 and PTK7 using γδ T cells, where CARs are separately encoded and dually expressed. Previously optimized transgenes containing GD2 or PTK7 scFv targeting domains followed by a CD8 hinge region, CD28 co-stim/trans-membrane domain, and CD3ζ signalling domain were inserted under the T7 promoter for mRNA production. Purified mRNA was electroporated into γδ T cells following their thaw from cryopreservation. Electroporation titrations were performed to optimize CAR expression to be detected up to 72 hours post-modification, which is compatible with the approximate in vivo lifespan γδ T cells in mice. Simultaneous expression of both CARs appears highest 24 h after electroporation, with ~70% of the target γδ T cell population modified. Anti-GD2/PTK7 γδ T cells are potent against the NB cell line IMR5 (GD2+PTK7+) in a 4 h cytotoxicity assay at effector:target ratios as low as 0.5:1. Importantly, specificity is also shown against NB cell lines genetically engineered to represent the clinical heterogeneity of GD2 and PTK7 expression that may be observed, where the dual CAR therapy is effective against GD2+PTK7+, GD2+PTK7−, and GD2−PTK7+, but not GD2−PTK7− NBs. In conclusion, we developed a dual CAR-based cellular therapy for NB using γδ T cells as an effector population in place of classical αβ T cells. Early studies demonstrate feasibility for innovative dual CAR expression and show promise for strong anti-NB potency. Future work will optimize CAR signaling domains for maximal efficacy in solid tumors, as well as confirm efficacy and safety in vivo with results rapidly translatable into our established γδ T cell clinical trial pipeline. Citation Format: Hunter C. Jonus, Jasmine Y. Lee, Jordan A. Silva, H. Trent Spencer, Kelly C. Goldsmith. Dual targeted CAR immunotherapy for neuroblastoma using γδ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4093.

Epigenetic therapies for neuroblastoma: immunogenicity awakens.

The development of immunotherapies for neuroblastoma remains challenging owing to the low immunogenicity of neuroblastoma cells, as reflected by the low expression of one of the main triggers of immune recognition, the major histocompatibility complex class I (MHC-I). Cornel et al. showed that epigenetic modulation of neuroblastoma cells with a histone deacetylase inhibitor can boost the expression of major histocompatibility complex class I, among other immune receptors, priming their recognition by T- and natural killer cells. By leveraging the developmentally related aberrant epigenetic landscapes of neuroblastoma, these discoveries pave the way to overcome a major limitation in the field of neuroblastoma immunotherapy.

Cytokine Immunotherapy for Neuroblastoma

Neuroblastoma (NB) is an embryonal tumor originating from the sympathetic nervous system, which occurs during fetal period or early postnatal period, and is one of the most common extracranial solid malignant tumors in children. Cytokines are pleiotropic proteins that can effectively activate tumor immune cells and counteract immune suppression, thereby suppressing tumors. Cytokine immunotherapy provides more possibilities for the application of immunotherapy through its own induction and activation of the immune system and has also been extensively studied in NB immunotherapy. This article mainly introduces the research progress of several immunotherapies based on cytokines in the treatment of children with NB.

Overexpression of lncRNA TUG1 enhances the efficacy of DC-CIK immunotherapy in neuroblastoma in vitro and in vivo.

Long non-coding RNA (LncRNA) TUG1 plays a critical role in the development of human cancers. This study explored whether TUG1 is involved in the cytotoxicity of dendritic cells and cytokine-induced killer cells (DCs-CIK), an immunotherapy approach, in neuroblastoma. A TUG1 expression plasmid was transfected into DCs. Neuroblastoma SK-N-SH cells were incubated with CIK cells, DCs-CIK cells, and TUG1-overexpressing DCs-CIK cells, with or without irradiation. SK-N-SH cell viability, colony formation, migration, and apoptosis were analyzed using CCK-8, colony formation assay, transwell assay, and flow cytometry, respectively. Production of IL-12, IL-2 and IFN-γ in the supernatants was determined using ELISA. A dual luciferase activity assay was performed to confirm the molecular interactions between TUG1 and miR-204. Tumor-bearing mice were established by injection of SK-N-SH cells followed by stimulation with CIK cells, DC-CIK cells, and TUG1-overexpressing DCs-CIK cells. Compared to CIK alone or DC-CIK therapy, overexpression of TUG1 significantly suppressed tumor cell proliferation, colony formation, and migration of neuroblastoma cells. Moreover, upregulation of TUG1 robustly induced apoptosis and altered key molecules associated with apoptosis and epithelial-mesenchymal transition. Contents of IL-12, IL-2 and IFN-γ were dramatically elevated in the supernatants in the coculturing system upon transfection with TUG1. In addition, TUG1 was found to be act as miR-204 sponge. Furthermore, in vivo experiments demonstrated that upregulation of TUG1 potentiated the antitumor activity of DC-CIK immunotherapy. Overexpression of TUG1 promotes DC maturation and enhances CIK cytotoxicity, suggesting that TUG1 may be a novel target for enhancing DC-CIK based immunotherapy for neuroblastoma.

A novel ganglioside-related risk signature can reveal the distinct immune landscape of neuroblastoma and predict the immunotherapeutic response.

Gangliosides play an essential role in cancer development and progression. However, the involvement of gangliosides in the prognosis and tumor microenvironment (TME) of neuroblastoma is not entirely understood. Consensus clustering analysis was performed to identify ganglioside-mediated molecular subtypes. LASSO-Cox analysis was conducted to identify independent prognostic genes, and a novel risk signature was constructed. The risk signature was validated internally and externally. We further explored the independent prognosis value, immune landscape, drug susceptibility, and tumor dedifferentiation of the risk signature. The role of the signature gene B3GALT4 in neuroblastoma was explored in vitro. Seventeen ganglioside-related genes were differentially expressed between INSS stage 4 and other stages, and two ganglioside-related clusters with distinct prognoses were identified. A novel risk signature integrating ten ganglioside-related prognostic genes was established. Across the train set and external validation sets, the risk signature presented high predictive accuracy and discrimination. The risk signature was an independent prognostic factor and constructed a nomogram combining multiple clinical characteristics. In the high-score group, the deficiency in antigen processing and presenting machinery, lack of immune cell infiltration, and escaping NK cells contributed substantially to immune escape. The low-score group was more responsive to immune checkpoint blockade therapy, while the high-score group showed substantial sensitivity to multiple chemotherapeutic drugs. Besides, the risk score was significantly positively correlated with the stemness index and reduced considerably in all-trans retinoic acid-treated neuroblastoma cell lines, indicating high dedifferentiation in the high-score group. Additionally, neuroblastoma cells with downregulation of B3GALT4 present with increased proliferation, invasion, and metastasis abilities in vitro. The novel ganglioside-related risk signature highlights the role of ganglioside in neuroblastoma prognosis and immune landscape and helps optimize chemotherapy and immunotherapy for neuroblastoma.

Expression of programmed cell death-1 on neuroblastoma cells in TH-MYCN transgenic mice.

Immunotherapy may improve the poor prognosis of high-risk neuroblastoma. Programmed cell death-1 (PD-1) is expressed in several cancers. The tyrosine hydroxylase MYCN (TH-MYCN) transgenic mouse model is widely used in neuroblastoma research, but detailed information on its immunological background is lacking. Therefore, we studied the immunological tumor microenvironment and tumor cell surface antigen expression in homozygote and hemizygote mice and effects of antibody therapy against PD-1. CD4, CD8, CD11b, and CD11c expression in immune cells from retroperitoneal lymph nodes and spleen was analyzed by flow cytometry. Tumor cell surface antigen expression was confirmed, and data from homozygote and hemizygote mice were compared. Effects of anti-PD-1 antibody were evaluated. CD4-, CD8-, CD11b-, and CD11c-positive cells were not significantly different in homozygote and hemizygote mice, and CD11b- and CD11c-positive cells were identified in the tumor microenvironment in both. Tumor cells expressed PD-1, and anti-PD-1 antibody had anti-tumor effects and significantly reduced the percentage of living tumor cells in cultures after 2h. The immunological background is similar in homozygote and hemizygote TH-MYCN transgenic mice, and both have PD-1-positive tumor cells. Anti-PD-1 antibody suppresses tumor growth. This mouse model may be a useful for studying immunotherapy of neuroblastoma.

A signature based on five immune-related genes to predict the survival and immune characteristics of neuroblastoma

BackgroundMYCN amplification (MNA) has been proved to be related to poor prognosis in neuroblastoma (NBL), but the MYCN-related immune signatures and genes remain unclear.MethodsEnrichment analysis was used to identify the significant enrichment pathways of differentially expressed immune-related genes (DEIRGs). Weight gene coexpression network analysis (WGCNA) was applied to reveal the correlation between these DEIRGs and MYCN status. Univariate and multivariate Cox analyses were used to construct risk model. The relevant fractions of immune cells were evaluated by CIBERSORT and single-sample gene set enrichment analysis (ssGSEA).ResultsFive genes, including CHGA, PTGER1, SHC3, PLXNC1, and TRIM55 were enrolled into the risk model. Kaplan–Meier survival analysis and receiver operating characteristic (ROC) curve showed that our model performed well in predicting the outcomes of NBL (3-years AUC = 0.720, 5-year AUC = 0.775, 10-years AUC = 0.782), which has been validated in the GSE49711 dataset and the E-MTAB-8248 dataset. By comparing with the tumor immune dysfunction and exclusion (TIDE) and tumor inflammation signature (TIS), we further proved that our model is reliable. Univariate and multivariate Cox regression analyses indicated that the risk score, age, and MYCN can serve as independent prognostic factors in the E-MATB-8248. Functional enrichment analysis showed the DEIRGs were enriched in leukocyte adhesion-related signaling pathways. Gene set enrichment analysis (GSEA) revealed the significantly enriched pathways of the five MYCN-related DEIRGs. The risk score was negatively correlated with the immune checkpoint CD274 (PD-L1) but no significant difference with the TMB. We also confirmed the prognostic value of our model in predicting immunotherapeutics.ConclusionWe constructed and verified a signature based on DEIRG that related to MNA and predicted the survival of NBL based on relevant immune signatures. These findings could provide help for predicting prognosis and developing immunotherapy in NBL.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

Use of anti-GD2 (Dinutuximab) as a target for CAR-T cells immunotherapy in neuroblastoma

Treating solid tumours with chimeric antigen receptors-modified T cells (CAR-T cells) has shown limited efficacy due to the lack of cancer-type specific antigens. Dinutuximab is a monoclonal antibody that recognizes a sphingolipid, disialoganglioside GD2, which has limited expression in normal tissues but is overexpressed in paediatric tumours, mainly neuroblastoma (NB) and diffuse intrinsic pontine glioma (DIPG) and other histone H3 K27M (H3K27M) mutated diffuse midline gliomas. Dinutuximab, an anti-GD2 antibody, is currently standard of care in the treatment of NB. We are contemplating the possibility of using Dinutuximab as target element for antiNB-CAR-T cells. Our group have explored the feasibility of producing CAR-T cells from peripheral blood (PB), post-apheresis CD45RA+ fraction and cord blood (CB) using similar protocols of T cell transduction and expansion ability as a way to favour the persistence of CAR-T cells in the host. Afterward, we performed a deeply characterization of phenotype and functionality of anti-FITC-CAR-T cells derived from different sources, and the cytotoxic effect against anti-GD2-FITC- NB labelled cell line. T cells purified from PB, CB and 45RA fraction after apheresis were cultured in vitro with cytokines IL-7+IL-15+IL- 21 to maintain early memory phenotypes as Naïve T cells (TN). A second generation lentiviral expression vector (LV) was used to generate second generation CARs (FITC-L-BBz CAR). After 48h of activation in culture with antiCD3/CD28 and cytokines, T cells were transduced with viral supernatants. Next, we performed a characterization of phenotype of anti-FITC-CAR-T cells by flow cytometry studying how the culture conditions enhanced a TN phenotype expressing CD45RA+ and CCR7+. Cytotoxic function of CAR-T cells was analysed in cocultures with LAN-1 NB cell line labelled with Dinutuximab conjugated with FITC. Preliminary results showed that culture condition with IL-7/15/21 maintained more primitive phenotypes in 45RA (71% TN CD4+ and 66% TN CD8+) and CB (60% of TN CD4+ and 77% of TN CD8+) derived T cells than PB T cells (only a 20% of TN CD4+ and TN CD8+). PB and CB-derived CAR-T cells showed higher cytolitic function than 45RA fraction, resulting in a 30% of dead cells in both cases in a 24h-culture assay. Both, PB and CB derived CAR-T cells showed the best CAR-T activation capacity with 19,6% of cells from PB and 33,9% of cells from CB expressing early activation markers as CD25 and CD134. In vivo experiments are currently on-going to test the efficacy of CAR-T cells in combination with GD2 treatment. Our strategy may complement the current use of Dinutuximab in the treatment of NB through its combination with a targeted CAR-T cell approach.

Immunotherapy of Neuroblastoma: Facts and Hopes.

While the adoption of multimodal therapy including surgery, radiation, and aggressive combination chemotherapy has improved outcomes for many children with high-risk neuroblastoma, we appear to have reached a plateau in what can be achieved with cytotoxic therapies alone. Most children with cancer, including high-risk neuroblastoma, do not benefit from treatment with immune checkpoint inhibitors (ICI) that have revolutionized the treatment of many highly immunogenic adult solid tumors. This likely reflects the low tumor mutation burden as well as the downregulated MHC-I that characterizes most high-risk neuroblastomas. For these reasons, neuroblastoma represents an immunotherapeutic challenge that may be a model for the creation of effective immunotherapy for other "cold" tumors in children and adults that do not respond to ICI. The identification of strong expression of the disialoganglioside GD2 on the surface of nearly all neuroblastoma cells provided a target for immune recognition by anti-GD2 mAbs that recruit Fc receptor-expressing innate immune cells that mediate cytotoxicity or phagocytosis. Adoption of anti-GD2 antibodies into both upfront and relapse treatment protocols has dramatically increased survival rates and altered the landscape for children with high-risk neuroblastoma. This review describes how these approaches have been expanded to additional combinations and forms of immunotherapy that have already demonstrated clear clinical benefit. We also describe the efforts to identify additional immune targets for neuroblastoma. Finally, we summarize newer approaches being pursued that may well help both innate and adaptive immune cells, endogenous or genetically engineered, to more effectively destroy neuroblastoma cells, to better induce complete remission and prevent recurrence.

Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy

BackgroundAnti-GD2 monoclonal antibody immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to treatment, and the molecular...

Dissecting the cellular components of ex vivo γδ T cell expansions to optimize selection of potent cell therapy donors for neuroblastoma immunotherapy trials

ABSTRACT γδ T lymphocytes represent an emerging class of cellular immunotherapy with preclinical promise to treat cancer, notably neuroblastoma. The innate-like immune cell subset demonstrates inherent cytoxicity toward tumor cells independent of MHC recognition, enabling allogeneic administration of healthy donor-derived γδ T cell therapies. A current limitation is the substantial interindividual γδ T cell expansion variation among leukocyte collections. Overcoming this limitation will enable realization of the full potential of allogeneic γδ T-based cellular therapy. Here, we characterize γδ T cell expansions from healthy adult donors and observe that highly potent natural killer (NK) lymphocytes expand with γδ T cells under zoledronate and IL-2 stimulation. The presence of NK cells correlates with both the expansion potential of γδ T cells and the overall potency of the γδ T cell therapy. However, the potency of the cell therapy in combination with an antibody-based immunotherapeutic, dinutuximab, appears to be independent of γδ T/NK cell content both in vitro and in vivo, which minimizes the implication of interindividual expansion differences toward efficacy. Collectively, these studies highlight the utility of maintaining the NK cell population within expanded γδ T cell therapies and suggest a synergistic action of combined innate cell immunotherapy toward neuroblastoma.