Shortened ex vivo manufacturing time of EGFRvIII-specific chimeric antigen receptor (CAR) T cells reduces immune exhaustion and enhances antiglioma therapeutic function.

BACKGROUND: Non-viral manufacturing of CAR T cells via the Sleeping Beauty transposon is cost effective and reduces the risk of insertional mutagenesis from viral transduction. However, the current gold standard methodology requires ex vivo numerical expansion of these cells on artificial antigen-presenting cells (AaPCs) for 4 weeks to generate CAR T cells of presumed sufficient quantity and function for clinical applications. METHOD: We engineered EGFRvIII-specific CAR T cells and monitored phenotypic changes throughout their ex vivo manufacturing. To reduce the culture time required to generate the CAR T-cell population, we selected for T cells in peripheral blood mononuclear cells prior to CAR modification (to eliminate the competing NK cell population). RESULTS: While we found increased expression of exhaustion markers (such as PD-1, PD-L1, TIM-3, and LAG-3) after 2 weeks in culture, whose levels continued to rise over time, we were able to generate a CAR+ T-cell population with comparable CAR expression and cell numbers in 2 weeks, thereby reducing manufacturing time by 50%, with lower expression of immune exhaustion markers. The CAR T cells manufactured at 2 weeks showed superior therapeutic efficacy in mice bearing established orthotopic EGFRvIII+ U87 gliomas. CONCLUSION: These findings demonstrate a novel, rapid method to generate CAR T cells by non-viral modification that results in CAR T cells superior in phenotype and function and further emphasizes that careful monitoring of CAR T-cell phenotype prior to infusion is critical for generating an optimal CAR T-cell product with full antitumor potential.

Symbiotic Macrophage-Glioma Cell Interactions Reveal Synthetic Lethality in PTEN-Null Glioma.

Heterotypic interactions across diverse cell types can enable tumor progression and hold the potential to expand therapeutic interventions. Here, combined profiling and functional studies of glioma cells in glioblastoma multiforme (GBM) models establish that PTEN deficiency activates YAP1, which directly upregulates lysyl oxidase (LOX) expression. Mechanistically, secreted LOX functions as a potent macrophage chemoattractant via activation of the ß1 integrin-PYK2 pathway in macrophages. These infiltrating macrophages secrete SPP1, which sustains glioma cell survival and stimulates angiogenesis. In PTEN-null GBM models, LOX inhibition markedly suppresses macrophage infiltration and tumor progression. Correspondingly, YAP1-LOX and ß1 integrin-SPP1 signaling correlates positively with higher macrophage density and lower overall survival in GBM patients. This symbiotic glioma-macrophage interplay provides therapeutic targets specifically for PTEN-deficient GBM.

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Mechanisms of action of rapamycin in gliomas.

Rapamycin has previously been shown to be efficacious against intracerebral glioma xenografts and to act in a cytostatic manner against gliomas. However, very little is known about the mechanism of action of rapamycin. The purpose of our study was to further investigate the in vitro and in vivo mechanisms of action of rapamycin, to elucidate molecular end points that may be applicable for investigation in a clinical trial, and to examine potential mechanisms of treatment failure. In the phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null glioma cell lines U-87 and D-54, but not the oligodendroglioma cell line HOG (PTEN null), doses of rapamycin at the IC50 resulted in accumulation of cells in G1, with a corresponding decrease in the fraction of cells traversing the S phase as early as 24 h after dosing. All glioma cell lines tested had markedly diminished production of vascular endothelial growth factor (VEGF) when cultured with rapamycin, even at doses below the IC50. After 48 h of exposure to rapamycin, the glioma cell lines (but not HOG cells) showed downregulation of the membrane type-1 matrix metalloproteinase (MMP) invasion molecule. In U-87 cells, MMP-2 was downregulated, and in D-54 cells, both MMP-2 and MMP-9 were downregulated after treatment with rapamycin. Treatment of established subcutaneous U-87 xenografts in vivo resulted in marked tumor regression (P < 0.05). Immunohistochemical studies of subcutaneous U-87 tumors demonstrated diminished production of VEGF in mice treated with rapamycin. Gelatin zymography showed marked reduction of MMP-2 in the mice with subcutaneous U-87 xenografts that were treated with rapamycin as compared with controls treated with phosphatebuffered saline. In contrast, treatment of established intracerebral U-87 xenografts did not result in increased median survival despite inhibition of the Akt pathway within the tumors. Also, in contrast with our findings for subcutaneous tumors, immunohistochemistry and quantitative Western blot analysis results for intracerebral U-87 xenografts indicated that there is not significant VEGF production, which suggests possible deferential regulation of the hypoxia-inducible factor 1alpha in the intracerebral compartment. These findings demonstrate that the complex operational mechanisms of rapamycin against gliomas include cytostasis, anti-VEGF, and anti-invasion activity, but these are dependent on the in vivo location of the tumor and have implications for the design of a clinical trial.