Generation of anti-GD2 CAR macrophages from human pluripotent stem cells for cancer immunotherapies
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PROVIDER: PRJNA828592 | ENA |
REPOSITORIES: ENA
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Project description:We generate macrophages from pluripotent stem cells (PSCs) with GD2 CARs integrated into AAVS1 locus. To produce CAR macrophages (CAR-M) we established a serum- and feeder-free differentiation protocol which generates macrophages through arterialized hemogenic endothelium.
2022-12-31 | GSE201174 | GEO
Project description:Vα24-invariant natural killer T cells (NKTs) have antitumor properties that can be enhanced by transgenic expression of tumor-specific receptors. Here, we report the results of the first-in-human clinical evaluation of autologous NKTs co-expressing a GD2-specific chimeric antigen receptor with interleukin (IL)15 (GD2-CAR.15) in 12 children with neuroblastoma (NB) treated on four dose levels (NCT03294954). Objectives included assessing safety, antitumor activity, and immune response. No dose-limiting toxicities occurred, and one patient had grade 2 cytokine release syndrome resolved by tocilizumab. The overall response rate was 25% (3/12) and disease control rate was 58% (7/12) including four patients with stable disease, two partial responses, and one complete response. CD62L+ NKT frequency in infused products correlated with CAR-NKT expansion in patients and was higher in responders than non-responders (71% vs 35.3%, p=0.002). Singe-cell RNA sequencing analysis identified B cell translocation gene 1 (BTG1) as one of the top upregulated genes in GD2-CAR.15-NKTs after in vitro serial tumor challenge. Genetic gain- and loss-of-function experiments revealed that BTG1 is a key driver of hyporesponsiveness in exhausted NKT and T cells. Crucially, NKTs co-expressing GD2-CAR.15 and BTG1-specific shRNA eradicated metastatic NB in mice. These results indicate that CAR-NKTs are safe, produce objective responses in NB patients, and that targeting BTG1 can enhance their therapeutic potency.
2023-04-19 | GSE223071 | GEO
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized the systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) between two generations of CAR-T cells aimed at integrating their fitness to overcome exhaustion and stress in tumor microenvironments Methods: mRNA profiles of CAR-T cells co-cultured with target cells for 48 hours were generated by deep sequencing. Human CD45 enrichment was performed to decrease tumor contamination. Results: Gene Set Enrichment Analysis (GSEA) of the RNA expression profile of T cells obtained 48 hours post co-culture with target cells demonstrated the enrichment of genes with memory T cell phenotype in B7H3 CAR-T compared to GD2-B7H3 T cells. However, B7H3 CAR-T cells were enriched in genes associated with glycolysis and apoptosis pathways suggesting a committed terminal fate for these T cells. There was significantly greater expression of genes attributed to exhaustion in B7H3 CAR-T cells than GD2-B7H3 T cells. In particular, B7H3 CAR-T cells showed higher expression of inhibitory receptors LAG3, HAVCR2 (TIM3), and BTLA genes, along with exhaustion-related transcription factor genes TBX21 (T-bet), PRDM1 (Blimp-1), and IKZF2 (Helios). B7H3 CAR-T cells also expressed genes that encode transcription factors reported to be associated with activated and memory T cells, such as KLF6, JUN, and JUNB. Conclusions: Our study represents the first analysis of transcriptome comparing SynNotch gated GD2-B7H3 CAR-T cells versus the conventional B7H3 CAR-T cells. GD2-B7H3 T cells exhibited superior resistance to exhaustion and greater metabolic fitness in comparison to conventional CAR-T cells. Metabolic preference (glycolytic versus oxidative) has an enormous impact on T cell fate. After eradicating NBL cells, GD2-B7H3 T cells had an oxygen consumption rate similar to UT cells, suggesting that the gated T cells can revert to their naïve metabolic state. Improved metabolic plasticity and reprogramming in favor of oxidative rather than glycolytic phosphorylation supports the hypothesis that the gated CAR-T cells likely have intact expansion potential, similar to unmanipulated naïve T cells. We observed significant enrichment of glycolytic genes in conventional B7H3 CAR-T cells compared to GD2-B7H3 T cells, supporting our metabolic studies. Our data suggest that SynNotch gated CAR-T cells have a more favorable oxidative metabolic profile than 4-1BB CAR-T cells.
2020-11-30 | GSE161942 | GEO
Project description:The purpose of the experiment was to compare the transcriptional profile of T-cells expressing a GD2-CAR construct or a GD2-CAR.C7R construct (where the GD2-CAR and the C7R construct are co-expressed), after the T-cells had been exposed to two serial tumor cell co-cultures. C7R is an engineered IL-7 receptor that has been rendered constitutively active. The sample replicates were generated using peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors.
2017-10-05 | GSE102567 | GEO
Project description:Purpose: To compare cell states between CD19-28z and GD2-28z human CAR T cells on day 10 of cell culture. Methods: Human T cells were activated and lentivirally transduced with CD19-28z or GD2-28z CAR constructs and maintained in culture for 10 days, and then delivered to the Stanford Functional Genomics Facility for 3' single-cell RNA-sequencing on the 10X Genomics platform. Results: Comparison of transcription factor profiles by single cell RNA-seq analysis of CD8+ T cells expressing CD19-28z vs. GD2-28z CAR confirmed that the bZIP family members JUN, JUNB, JUND, and ATF4 were among the most differentially expressed and broadly connected in exhausted GD2-28z CAR T cells. Conclusions: This study provides insights into cell states that could explain the underlying differences between highly functional CD19-28z CAR T cells and exhaustion-prone GD2-28z CAR T cells on day 10 in culture.
2019-10-01 | GSE136874 | GEO
Project description:Vα24-invariant natural killer T cells (NKTs) have antitumor properties that can be enhanced by transgenic expression of tumor-specific receptors. Here, we report the results of the first-in-human clinical evaluation of autologous NKTs co-expressing a GD2-specific chimeric antigen receptor with interleukin (IL)15 (GD2-CAR.15) in 12 children with neuroblastoma (NB) treated on four dose levels (NCT03294954). Objectives included assessing safety, antitumor activity, and immune response. No dose-limiting toxicities occurred, and one patient had grade 2 cytokine release syndrome resolved by tocilizumab. The overall response rate was 25% (3/12) and disease control rate was 58% (7/12) including four patients with stable disease, two partial responses, and one complete response. CD62L+ NKT frequency in infused products correlated with CAR-NKT expansion in patients and was higher in responders than non-responders (71% vs 35.3%, p=0.002). Singe-cell RNA sequencing analysis identified B cell translocation gene 1 (BTG1) as one of the top upregulated genes in GD2-CAR.15-NKTs after in vitro serial tumor challenge. Genetic gain- and loss-of-function experiments revealed that BTG1 is a key driver of hyporesponsiveness in exhausted NKT and T cells. Crucially, NKTs co-expressing GD2-CAR.15 and BTG1-specific shRNA eradicated metastatic NB in mice. These results indicate that CAR-NKTs are safe, produce objective responses in NB patients, and that targeting BTG1 can enhance their therapeutic potency.
2023-05-01 | GSE220927 | GEO
Project description:Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal central nervous system (CNS) tumors that occur most commonly in children and young adults1. Average life expectancy is ten months from diagnosis, and 5-year survival is less than 1%2. Palliative radiotherapy is the only established treatment3, and neither cytotoxic nor targeted pharmacological approaches have demonstrated anti-tumor responses or improved prognosis to date3,4. We previously discovered that the disialoganglioside GD2 is highly and uniformly expressed on H3K27M+ DMG cells and demonstrated that intravenously (IV) administered GD2.4-1BB.z chimeric antigen receptor (CAR) T-cells eradicated established DMGs in patient-derived orthotopic murine models5, thereby providing the rationale for a first-in-human/first-in-child Phase 1 clinical trial (NCT04196413). Because CAR T-cell-induced inflammation and edema of the brainstem can result in obstructive hydrocephalus, increased intracranial pressure, and dangerous tissue shifts, a number of neurocritical care precautions were incorporated in the clinical trial design and management plan. Here we present the clinical experience from the first four patients with H3K27M+ DMG treated with GD2-CAR T-cells at dose level 1 (DL1; 1e6 GD2-CAR T-cells/kg administered IV). Patients who exhibited clinical benefit were eligible for subsequent administrations of GD2-CAR T-cells. Given preclinical evidence for increased CAR T-cell potency6, and the potential for diminished immunogenicity with locoregional administration, second doses were administered intracerebroventricularly (ICV) through an Ommaya catheter to three patients. As predicted from preclinical models, toxicity was largely related to the neuroanatomical location of the tumors and was reversible with intensive supportive care. Although GD2 is expressed at low levels in normal neural tissue, no evidence of on-target, off-tumor toxicity was observed. Three of four patients exhibited clinical and radiographic improvement, underscoring the promise of this approach for H3K27M+ DMG therapy. Correlative studies of serum and CSF revealed marked proinflammatory cytokine production following GD2 CAR T cell administration and single cell transcriptomic analysis of 65,598 single cells from CAR T cell products and patient CSF has begun to reveal differences that correlate with the heterogeneity between subjects and routes of administration.
2022-02-07 | GSE186802 | GEO
Project description:In this work, we compared the expression profiles of Anti-GD2- transduced T-cells with Anti-GD2-transduced T-cells co-cultured with SK-Mel-37.
2023-07-31 | GSE238193 | GEO
Project description:Immunotranscriptomic Study of Human anti-GD2 CAR T-cells
| PRJNA998574 | ENA
Project description:GD2 is a disialoganglioside that is highly expressed on the surface of neuroblastoma cells. Immunotherapy with anti-GD2 antibodies has revolutionized the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse and little is known about mechanisms of resistance to anti-GD2. Neuroblastomas harbor intrinsic transcriptional plasticity by co-opting divergent lineage-specific developmental programs between adrenergic and mesenchymal cell states. We found that reduced GD2 expression was significantly correlated with the adrenergic cell state in neuroblastoma and that an Adrenergic-to-Mesenchymal Transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Induced reprogramming of adrenergic cells with the master AMT regulator PRRX1 was sufficient to promote transcriptional rewiring in isogenic models and downregulate GD2 expression. Mechanistically, low-GD2 expressing cell lines demonstrate significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Primary neuroblastoma tumors enriched for mesenchymal features show demonstrably lower GD3 synthase expression as compared to adrenergic tumors. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to an anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.
2022-04-26 | GSE196861 | GEO