Armored bicistronic CAR T cells with dominant-negative TGF-ß receptor II to overcome resistance in glioblastoma.

Chimeric antigen receptor (CAR) T cells have shown significant efficacy in hematological diseases. However, CAR T therapy has demonstrated limited efficacy in solid tumors, including glioblastoma (GBM). One of the most important reasons is the immunosuppressive tumor microenvironment (TME), which promotes tumor growth and suppresses immune cells used to eliminate tumor cells. The human transforming growth factor ß (TGF-ß) plays a crucial role in forming the suppressive GBM TME and driving the suppression of the anti-GBM response. To mitigate TGF-ß-mediated suppressive activity, we combined a dominant-negative TGF-ß receptor II (dnTGFßRII) with our previous bicistronic CART-EGFR-IL13Rα2 construct, currently being evaluated in a clinical trial, to generate CART-EGFR-IL13Rα2-dnTGFßRII, a tri-modular construct we are developing for clinical application. We hypothesized that this approach would more effectively subvert resistance mechanisms observed with GBM. Our data suggest that CART-EGFR-IL13Rα2-dnTGFßRII significantly augments T cell proliferation, enhances functional responses, and improves the fitness of bystander cells, particularly by decreasing the TGF-ß concentration in a TGF-ß-rich TME. In addition, in vivo studies validate the safety and efficacy of the dnTGFßRII cooperating with CARs in targeting and eradicating GBM in an NSG mouse model.

Brain-wide neuronal circuit connectome of human glioblastoma.

Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression 1-6 . Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic 7-9 . The extent of integration of GBM cells into brain-wide neuronal circuitry is not well understood. Here we applied a rabies virus-mediated retrograde monosynaptic tracing approach 10-12 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrated into brain-wide neuronal circuits and exhibited diverse local and long-range connectivity. Beyond glutamatergic inputs, we identified a variety of neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. Acute acetylcholine stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via the metabotropic CHRM3 receptor. CHRM3 downregulation suppressed GBM cell invasion, proliferation, and survival in vitro and in vivo. Together, these results reveal the capacity of human GBM cells to rapidly and robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain and support a model wherein rapid synapse formation onto GBM cells and transient activation of upstream neurons may lead to a long-lasting increase in fitness to promote tumor infiltration and progression.

Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results.

Recurrent glioblastoma (rGBM) remains a major unmet medical need, with a median overall survival of less than 1 year. Here we report the first six patients with rGBM treated in a phase 1 trial of intrathecally delivered bivalent chimeric antigen receptor (CAR) T cells targeting epidermal growth factor receptor (EGFR) and interleukin-13 receptor alpha 2 (IL13Rα2). The study's primary endpoints were safety and determination of the maximum tolerated dose. Secondary endpoints reported in this interim analysis include the frequency of manufacturing failures and objective radiographic response (ORR) according to modified Response Assessment in Neuro-Oncology criteria. All six patients had progressive, multifocal disease at the time of treatment. In both dose level 1 (1 ×107 cells; n = 3) and dose level 2 (2.5 × 107 cells; n = 3), administration of CART-EGFR-IL13Rα2 cells was associated with early-onset neurotoxicity, most consistent with immune effector cell-associated neurotoxicity syndrome (ICANS), and managed with high-dose dexamethasone and anakinra (anti-IL1R). One patient in dose level 2 experienced a dose-limiting toxicity (grade 3 anorexia, generalized muscle weakness and fatigue). Reductions in enhancement and tumor size at early magnetic resonance imaging timepoints were observed in all six patients; however, none met criteria for ORR. In exploratory endpoint analyses, substantial CAR T cell abundance and cytokine release in the cerebrospinal fluid were detected in all six patients. Taken together, these first-in-human data demonstrate the preliminary safety and bioactivity of CART-EGFR-IL13Rα2 cells in rGBM. An encouraging early efficacy signal was also detected and requires confirmation with additional patients and longer follow-up time. ClinicalTrials.gov identifier: NCT05168423 .

Integrating imaging and genomic data for the discovery of distinct glioblastoma subtypes: a joint learning approach.

Glioblastoma is a highly heterogeneous disease, with variations observed at both phenotypical and molecular levels. Personalized therapies would be facilitated by non-invasive in vivo approaches for characterizing this heterogeneity. In this study, we developed unsupervised joint machine learning between radiomic and genomic data, thereby identifying distinct glioblastoma subtypes. A retrospective cohort of 571 IDH-wildtype glioblastoma patients were included in the study, and pre-operative multi-parametric MRI scans and targeted next-generation sequencing (NGS) data were collected. L21-norm minimization was used to select a subset of 12 radiomic features from the MRI scans, and 13 key driver genes from the five main signal pathways most affected in glioblastoma were selected from the genomic data. Subtypes were identified using a joint learning approach called Anchor-based Partial Multi-modal Clustering on both radiomic and genomic modalities. Kaplan-Meier analysis identified three distinct glioblastoma subtypes: high-risk, medium-risk, and low-risk, based on overall survival outcome (p < 0.05, log-rank test; Hazard Ratio = 1.64, 95% CI 1.17-2.31, Cox proportional hazard model on high-risk and low-risk subtypes). The three subtypes displayed different phenotypical and molecular characteristics in terms of imaging histogram, co-occurrence of genes, and correlation between the two modalities. Our findings demonstrate the synergistic value of integrated radiomic signatures and molecular characteristics for glioblastoma subtyping. Joint learning on both modalities can aid in better understanding the molecular basis of phenotypical signatures of glioblastoma, and provide insights into the biological underpinnings of tumor formation and progression.

Live Organoid Cyclic Imaging.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab show no efficacy in glioblastoma: a phase 1 trial.

We previously showed that chimeric antigen receptor (CAR) T-cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces upregulation of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Here we conducted a phase 1 trial (NCT03726515) of CAR T-EGFRvIII cells administered concomitantly with the anti-PD1 (aPD1) monoclonal antibody pembrolizumab in patients with newly diagnosed, EGFRvIII+ glioblastoma (GBM) (n = 7). The primary outcome was safety, and no dose-limiting toxicity was observed. Secondary outcomes included median progression-free survival (5.2 months; 90% confidence interval (CI), 2.9-6.0 months) and median overall survival (11.8 months; 90% CI, 9.2-14.2 months). In exploratory analyses, comparison of the TME in tumors harvested before versus after CAR + aPD1 administration demonstrated substantial evolution of the infiltrating myeloid and T cells, with more exhausted, regulatory, and interferon (IFN)-stimulated T cells at relapse. Our study suggests that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicates a need to consider alternative strategies.