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GPC2-CAR T-cells have potent preclinical activity against orthotopic medulloblastoma xenografts

ABSTRACT: Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients with high-risk features, such as MYCN aberrations, and relapsed/refractory disease have poor clinical outcomes. This underscores the urgent need for new therapies. Glypican-2 (GPC2) is a MYCN-regulated and recently discovered oncofetal antigen. Given that GPC2 is also expressed in brain tumors, we evaluated the preclinical activity of our GPC2-CAR (CT3-CD28HTM-BBζ) against MB and compared it to two existing CARs targeting GD2 and B7-H3. A newly generated patient-derived xenograft (PDX) MAF1433, with MYCN aberrations, were used to test CAR T-cells in vivo. Single-cell RNA-sequencing were used for mechanistic studies. MB patient samples express intermediate to high levels of GPC2 and can be targeted with a GPC2-CAR, leading to significant in vivo tumor regression in orthotopic tumor models. GPC2-CAR T-cells had equivalent activity to the B7-H3-CAR and enhanced activity compared to the GD2-CAR in vivo. T-cell kinetic studies revealed that GPC2-CAR T-cells home to the TME, expand, and upregulate genes critical for cytotoxicity and T-cell homing to induce MB cell death. CT3-CD28HTM-BBζ GPC2-CAR can regress GPC2+ MB with MYCN aberrations in preclinical studies, providing a preclinical rationale for including children with GPC2+ MB in our upcoming clinical GPC2-CAR T-cell trial.

ORGANISM(S): Homo sapiens/Mus musculus xenograft

PROVIDER: GSE281233 | GEO | 2025/11/06

REPOSITORIES: GEO

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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.

Project description:BACKGROUND Focal high-level amplifications of MYC define a subset of high-risk medulloblastoma patients. However, the prognostic role of MYCN oncogene amplification remains less clear. We aimed to evaluate the prognostic value of this alteration alone and in combination with biological modifiers in a large cohort of 67 pediatric medulloblastomas with MYCN amplification (MYCN-MB). METHODS Twenty-one MYCN-MB with MYCN amplication and 56 MYCN-MB were respectively examined using either gene expression profiling and array-CGH, or immunohistochemical analysis and FISH. All 67 tumors were further subject to mutational analyses. Semi non-negative matrix factorization–based and unsupervised hierarchical clustering methods were used to identify biological groups. We compared molecular, clinical, and prognostic characteristics both within biological MYCN-MB groups and with non-amplified tumors. RESULTS Transcriptomic analysis of this large cohort of MYCN-MBs demonstrated a significant enrichment of MYCN-MB in the SHH and group D variants. Further substantiating this biological dichotomy of MYCN-MB, respective group affiliations were also accompanied by variant-specific cytogenetic aberrations including deletion of 9q in the SHH variant, and gain of 7q and isochromosome 17q/17q gain in MYCN-MBs clustering with group D tumors. Among clinically relevant variables, SHH subtype and 10q loss for Non-SHH tumors comprised the most powerful markers of favorable prognosis in MYCN-MB. CONCLUSION We demonstrate considerable heterogeneity within MYCN-MB in terms of genetics, tumor biology, and clinical outcome. Thus, assessment of disease group and 10q copy-number status may improve risk stratification of this group and may delineate MYCN-MB with the same dismal prognosis as MYC amplified tumors. Furthermore, based on the enrichment of MYCN and GLI2 amplifications in SHH-driven medulloblastoma, amplification of these downstream signaling intermediates should be excluded before a patient is enrolled into a clinical trial using a Smoothened inhibitor.

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GPC2-CAR T-cells have potent preclinical activity against orthotopic medulloblastoma xenografts

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