Glioblastoma multiforme (GBM) remains refractory to current treatment modalities. Differentiation-based approaches, which force cancer stem/progenitor cells to exit the cell cycle and adopt terminal fates, offer an alternative therapeutic strategy. Cell fate regulators operating during stem/progenitor cell divisions integrate proliferative and anti-proliferative cues and represent particularly attractive points of intervention. Here, we investigated the therapeutic potential of targeting mitotic kinesin KIF20A in GBM stem/progenitor cells. KIF20A is a crucial component of cytokinetic machinery and cooperates with a network of cell fate regulators to balance proliferative and differentiative divisions in neural stem/progenitor cells (NSPCs). Using complementary in vitro and in vivo models, including 2D cultures, 3D organoids, and intracranial xenografts, we show that inhibition of KIF20A drives cell cycle exit and induces a postmitotic/differentiated state in GBM stem/progenitor cells, resulting in a marked suppression of proliferation. Together, these findings establish KIF20A as a key vulnerability in GBM and a promising target for differentiation-based intervention.

Gliomas are among the most lethal cancers, with limited treatment options. To uncover hallmarks of therapeutic escape and tumor microenvironment (TME) landscape, we applied spatial proteomics, transcriptomics, and glycomics to 670 lesions from 310 adult and pediatric patients. Single-cell analysis shows high B7H3+ tumor cell prevalence in glioblastoma (GBM) and pleomorphic xanthoastrocytoma, while most gliomas, including pediatric cases, express targetable tumor antigens in less than 50% of tumor cells, potentially explaining trial failures. Paired samples of isocitrate dehydrogenase (IDH)-mutant gliomas reveal recurrence driven by tumor-immune spatial reorganization, shifting from T cell and vasculature-associated myeloid cell-enriched niches to microglia and CD206+ macrophage-dominated tumors. Multi-omic integration identified N-glycosylation as the best classifier of grade, while the immune transcriptome best predicted GBM survival. Provided as a community resource, this study offers a framework for glioma targeting, classification, outcome prediction, and a baseline of TME composition across all stages.

A challenge in treating glioblastoma (GBM) is its phenotypic heterogeneity between patients and within tumors. Chlorotoxin (CLTX), a peptide from scorpion venom, broadly binds glioma cells through a mechanism involving surface matrix metalloproteinase-2 (MMP-2). We previously developed chimeric antigen receptor (CAR) T cells incorporating CLTX as the GBM recognition domain. Here, we report interim clinical experience of a phase 1 trial evaluating intracavity/intratumoral (ICT) delivery of CLTX-CAR T cells in four patients with MMP-2-expressing recurrent GBM (NCT04214392), with the primary objectives of feasibility and safety. The therapy is well tolerated with no dose-limiting toxicities. Three of the four participants (75%) exhibit a best response of stable disease. CLTX-CAR T cells are detected in the tumor cavity fluid and at lower levels in the blood. Human anti-CAR antibody assays do not detect humoral immunogenicity against the CLTX-CAR. These observations support further clinical evaluation of CLTX-CAR therapy.

A promising advance for ex vivo studies of human brain development and formulation of therapeutic strategies has been the adoption of brain organoids that, to a greater extent than monolayer or spheroid cultures, recapitulate to varying extents the patterns of tissue development and cell differentiation of human brain. Previously, such studies been hampered by limited access to relevant human tissue, inadequate human in vitro models, and the necessity of using rodent models that imperfectly reproduce human brain physiology. Here we present a novel organoid-based research platform utilizing L-MYC-immortalized human fetal neural stem cells (LMNSC01) grown in a physiological 4% oxygen environment. We visualized developmental processes in LMNSC01 brain organoids for over 120 days in vitro by immunofluorescence and NanoString gene expression profiling. Gene expression patterns revealed by NanoString profiling were quantitatively compared to those occurring during normal brain development (BrainSpan database) using the Singscore method. We observe similar developmental patterns in LMNSC01 organoids and developing cortex for genes characterizing neurons, astrocytes, and oligodendrocytes, and multiple pathways including those involved in apoptosis, neuronal cytoskeleton, neurotransmission, and metabolism. Notable properties of this LMNSC01 platform are its initiation with immortalized authentic human neural stem cells, growth in a physiological oxygen environment, the consistency of the organoids produced, and favorable comparison of their gene expression patterns with those reported for normal cortical development.

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 106 CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .

Figure S1. Effect of JMJD3 on nuclear architecture.

Background IgE-producing multiple myeloma, non-Hodgkin’s lymphoma and chronic B cell leukemia are caused by neoplasm of IgE-producing B cells. Several chimeric antigen receptor (CAR) T-cell therapies have been approved for treating B cell malignancies, but they target B cell linage markers CD19 or BCMA and indiscriminately eliminate both malignant B cells and normal B cells. This leads to general B cell and plasma aplasia and compromised humoral immune responses. Methods To specifically target IgE-producing cancer cells, we developed a CAR that specifically recognizes the transmembrane form of IgE (mIgE). The second-generation CAR employs a single chain variable fragment (scFv) 2E3E10 that recognizes the extracellular membrane-proximal domain (EMPD), which is found only on mIgE and not on secreted IgE. To determine the activity of EMPD-specific CAR, U-266 myeloma cells expressing low levels of mIgE and Daudi lymphoma cells expressing high levels of mIgE were used as target cells. To minimize T cell receptor (TCR)-mediated alloreactive killing that is independent of CAR activity, beta-2-microglobulin (β2m) expression on U-266 cells were knocked out using CRISPR/Cas9 to abrogate HLA class I expression. In addition, U-266 and Daudi cells were modified to express firefly luciferase for monitoring target cell killing in vitro and in vivo. Results Primary human CD8+ and CD4+ T cells transduced using lentiviral vectors encoding the CAR showed high levels of CAR expression on around 50% of cells for more than three weeks in culture. Coculturing U-266-b2mKO-luci or Daudi-mIgE-luci target cells with the CAR T cells led to significant T cell cytokine production and target cell killing, demonstrating the ability of EMPD-specific CAR to mediate primary human T cell activation and cytotoxicity. To assess CAR T cell activity in vivo, myeloma and lymphoma mouse models were established through intravenous injection of U-266-b2mKO-luci and Daudi-mIgE-luci cells, respectively, into immunocompromised NSG mice. Administration of EMPD-specific CAR T cells three days later led to complete elimination of cancer cells in both xenograft mouse models. EMPD-specific CAR-T cells were detected in tail vein blood after 15 weeks and displayed stem and memory-like phenotypes. Conclusions Our results indicate that EMPD-CAR T cells are capable of mediating primary human T cell activation and cytotoxicity through recognition of the mIgE on IgE-expressing cancer cells both in vitro and in vivo, demonstrating the proof-of-concept for using EMPD-CAR T cells to treat IgE-producing blood cancers without the side effect of causing B cell aplasia.

Figure S2. (A) U251.JMJD3wt cells lack γH2AX foci. U251.JMJD3wt cells immunostained for γH2AX foci (red) and counterstained with DAPI (blue). As a positive control for γH2AX foci formation, U251.JMJD3wt cells were irradiated (Cs137 source) with 10 Gy γ-radiation and examined after 24 hr. Note the absence of γH2AX foci in untreated U251.JMJD3wt cells. Scale bar = 10 µm. (B) Western blot for γH2AX in U251.JMJD3 cells showing the relative deficit of γH2AX in non-irradiated cells.

Supplementary Figure S4. 18F-Fluorodeoxyglucose ( F-FDG)-CT/PET of subjects UPN031 and UPN033

Table S1. Ratios (U251.JMJD3wt vs. vector control) and p-values for 37,814 transcripts and 20,661 genes (duplicate transcripts removed) affected by JMJD3 over-expression in U251 cells.

Supplementary Figure S1. Catheter position with respect to resection site.

Supplementary Figure S5. T cell detection in tumor tissue from UPN031.

Background Chimeric antigen receptor (CAR) T cell therapy is being explored in early-stage clinical trials as a strategy to improve treatment outcomes for high-grade gliomas (HGGs). We report here, a completed phase I trial (NCT02208362) evaluating locoregionally delivered IL13Rα2-targeted CAR T cells in 65 patients with recurrent HGG (rHGG), the majority being recurrent glioblastoma (rGBM). Methods This five-arm trial evolved to evaluate three routes of locoregional CAR T cell administration: (i) intratumoral (ICT) following tumor biopsy (Arm 1) or resection (Arm 2); (ii) intraventricular (ICV; Arm 3); and (iii) dual ICT/ICV (Arm 4). The final treatment arm (Arm 5) evaluated dual ICT/ICV delivery with a modified manufacturing process. Primary objectives were feasibility and safety. Secondary objectives evaluated therapy-related cytokine dynamics by cytometric bead array, CAR T cell persistence by flow cytometry and PCR, and clinical outcomes by radiographic imaging and survival. The pretreatment tumor immune landscape was evaluated by immunohistochemistry. Results Feasibility and safety were established for all three routes of locoregional CAR T cell delivery (ICT, ICV and dual ICT/ICV). IL13Rα2-CAR T cells were well-tolerated with clinically manageable adverse events at all dose levels, and no dose limiting toxicities observed. Stable disease or better was achieved in 50% of patients, with two partial responses, one complete response (CR), and a second CR after additional CAR T cycles off protocol therapy. Median overall survival (OS) for rGBM patients (68% treated at 2nd recurrence or later) was 7.7 mo. Post-hoc analysis revealed that Arm 5 rGBM patients exhibited the best median OS of 10.2 months, compared to 6.1 months for other treatment arms (Arms 1–4). Increase in inflammatory cytokines, including IFNγ, CXCL9, and CXCL10, was observed in the cerebrospinal (CSF) and tumor fluid after each infusion. Further, pre-treatment intratumoral CD3 T cell levels were positively associated with survival. Conclusions We report the largest CAR T cell clinical trial in brain tumors to date, assessing the feasibility, safety, and bioactivity of IL13Rα2-CAR T cells in rHGG. Key findings include: (1) repetitive locoregional administration of IL13Rα2-CAR T cells is feasible and safe, with clinical benefit observed in a subset of patients; (2) elevations in IFNγ-related chemokines in the CSF was associated with CAR T cell administration and bioactivity; and (3) tumor immune contexture was identified as a determinant of patient outcome to CAR T cell therapy. These findings advance our understanding of CAR T cell immunotherapy for malignant brain tumors. Ethics Approval This study was conducted I accordance with the Institutional Review Board and Independent Ethics Committee at The City of Hope National Medical Center as well as the U.S. Food and Drug Administration (FDA). All subjects provided written informed consent.

Supplementary methods. These are more detailed methods than can be included in the body of the article.

Supplementary Figure S2. Consort flow diagram.

Supplementary Tables S1-3. Table S1. T cell Product Release Criteria. Table S2. Manufacturing Feasibility. Table S3. Characteristics of Patients Who Received Study Treatment.