Carley Fowler

Abstract H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the disialoganglioside GD2 (ref. 1 ). Chimeric antigen receptor-modified T cells targeting GD2 (GD2-CART) eradicated DMGs in preclinical models 1 . Arm A of Phase I trial no. NCT04196413 (ref. 2 ) administered one intravenous (IV) dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal DMG (sDMG) at two dose levels (DL1, 1 × 10 6 kg − 1 ; DL2, 3 × 10 6 kg −1 ) following lymphodepleting chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) intracranial infusions (10–30 × 10 6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability and the identification of maximally tolerated IV dose. Secondary objectives included preliminary assessments of benefit. Thirteen patients enroled, with 11 receiving IV GD2-CART on study ( n = 3 DL1 (3 DIPG); n = 8 DL2 (6 DIPG, 2 sDMG)). GD2-CART manufacture was successful for all patients. No dose-limiting toxicities occurred on DL1, but three patients experienced dose-limiting cytokine release syndrome on DL2, establishing DL1 as the maximally tolerated IV dose. Nine patients received ICV infusions, with no dose-limiting toxicities. All patients exhibited tumour inflammation-associated neurotoxicity, safely managed with intensive monitoring and care. Four patients demonstrated major volumetric tumour reductions (52, 54, 91 and 100%), with a further three patients exhibiting smaller reductions. One patient exhibited a complete response ongoing for over 30 months since enrolment. Nine patients demonstrated neurological benefit, as measured by a protocol-directed clinical improvement score. Sequential IV, followed by ICV GD2-CART, induced tumour regressions and neurological improvements in patients with DIPG and those with sDMG.

CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

Abstract The intensive nutrient requirements needed to sustain T cell activation and proliferation, combined with competition for nutrients within the tumor microenvironment, raise the prospect that glucose availability may limit CAR-T cell function. Here, we seek to test the hypothesis that stable overexpression (OE) of the glucose transporter GLUT1 in primary human CAR-T cells would improve their function and antitumor potency. We observe that GLUT1OE in CAR-T cells increases glucose consumption, glycolysis, glycolytic reserve, and oxidative phosphorylation, and these effects are associated with decreased T cell exhaustion and increased Th 17 differentiation. GLUT1OE also induces broad metabolic reprogramming associated with increased glutathione-mediated resistance to reactive oxygen species, and increased inosine accumulation. When challenged with tumors, GLUT1OE CAR-T cells secrete more proinflammatory cytokines and show enhanced cytotoxicity in vitro, and demonstrate superior tumor control and persistence in mouse models. Our collective findings support a paradigm wherein glucose availability is rate limiting for effector CAR-T cell function and demonstrate that enhancing glucose availability via GLUT1OE could augment antitumor immune function.

BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

Abstract Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8 + CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing. Statement of Significance Adenosine is well known to inhibit T cell function and substantial effort has focused on inhibiting adenosine generation and signaling. Here, we show that exhausted T cells are suppressed by adenosine, which is only modestly impacted by inhibiting adenosine generation or signaling. In contrast, metabolism of adenosine to inosine augmented T cell function and culture of T cells with inosine induced multi-level reprogramming leading to stemness and improved anti-tumor potency. We demonstrate the feasibility of introducing inosine during GMP cell manufacturing as a novel strategy to generate enhanced CAR-T cells.