Remote Metronomic Control of CAR T Cells to Overcome Brain Tumor Immunosuppression

Introduction: : Glioblastoma (GBM) remains one of the most difficult cancers to treat, in part due to the immunosuppressive brain tumor microenvironment. Myeloid-derived suppressor cells (MDSCs) are a dominant component of this suppressive niche, limiting CAR T cell activation, expansion, and cytotoxicity. Brain tumors also present a spatially constrained therapeutic window where systemically delivered agents pose significant toxicity risks and lack precision. Administration of biologics, such as monoclonal antibodies, small molecules, and bispecific T cell engagers (BTEs), are being investigated to improve clinical efficacy of immunotherapies yet are limited by their dose-limiting toxicities, poor penetration across the blood-brain barrier, and rapid clearance from sites of disease. We reasoned that engineered T cells capable of producing immunomodulatory payloads transiently and locally could overcome these barriers. Leveraging the success of chimeric antigen receptor (CAR) T cells for treating hematological malignancies, we aim to enhance delivery and safety in treating BCBM patients by incorporating potent immunostimulatory agents such as cytokines (e.g., IL-2), checkpoint blockade inhibitor (e.g., anti-PD-1), and T cell engagers (TCEs). Here, we describe a sonogenetic platform that enables closed-loop, metronomic control of CAR T cell function in the brain using MRI-guided focused ultrasound (MRgFUS). T cells are engineered with a thermal gene switch to produce murine NKG2D bispecific T cell engagers (TS.mTCEs), allowing remotely controlled engagement of endogenous T cells against suppressive myeloid populations found in GBM. This design enables localized immune remodeling without the need for continuous transgene expression or systemic exposure.

Materials and

Methods: : To assess the ability of the thermal switch to drive transient and repeatable transgene expression, 5×10⁵ primary human T cells engineered with a thermally inducible firefly luciferase reporter (TS.Fluc) were administered intratumorally into mice bearing brain tumors. Tumors were selectively heated to 41.5°C for 10 minutes using a closed-loop, PID-controlled MR-guided focused ultrasound (MRgFUS) system (Fig. 1a). Luminescent imaging (IVIS) was performed 10 hours post-heating to quantify transgene activation. To evaluate the effects of local TCE production on immune remodeling in GBM tumors, we implanted 2×103 SB28 murine glioblastoma cells intracranially into C57BL/6 mice. 8 days after inoculation, mice received 2.5×104 murine T cells transduced to express TS.mTCE upstream of EGFRvIII CAR via intratumoral injection. MRgFUS was used to deliver localized mild hyperthermia (41°C for 10 minutes) to the tumor 1 day, 6 days, and 11 days following ACT guided by T2-weighted MRI and verified with MR thermometry. Metronomic dosing was used to allow transgene reset between pulses while enabling sustained immunomodulation throughout the duration of treatment and mitigate off-target toxicity or exhaustion (Fig. 1d). After a single thermal treatment (3 days post ACT) mice were sacrificed for flow cytometry. Brain hemispheres were dissociated into single-cell suspensions and stained to quantify CD45, CAR expression, and MDSC subsets (CD11b⁺Ly6C⁺Ly6G⁻). To assess therapeutic impact, tumor burden was measured by IVIS bioluminescence imaging throughout the duration of treatment.

Results, Conclusions, and Discussions:: We sought to determine whether remote, metronomic activation of engineered CAR T cells could reshape the immune landscape in brain tumors. Following MRgFUS mediated heating, luminescence imaging revealed a ~10 fold increase in reporter expression within tumors compared to unheated controls (Fig. 1b). Given the transient nature of the thermal bioswitch, and the safety of the MRgFUS-mediated heating, we applied a second round of sonothermal activation, inducing another peak in transgene expression, demonstrating the feasibility of in vivo metronomic dosing (Fig. 1c). To evaluate the effect of local TCE production on the immune environment of murine GBM, mice bearing orthotopic SB28-EGFRvIII tumors received TS.mTCE CAR T cells intratumorally and underwent three cycles of spatially confined heating using MRgFUS (Fig. 1d). Flow cytometry revealed no difference in the number or frequency of CAR T cells in the tumor between heated and unheated groups (Fig. 1e). However, MRgFUS-treated mice showed a significant reduction in MDSCs (CD11b⁺Ly6C⁺Ly6G⁻), both in frequency and total number following a single thermal treatment (Fig. 1f). In a longitudinal study to assess therapeutic efficacy, WT T cells had no significant effect on tumor burden regardless of whether FUS treatment was used in combination, confirming that hyperthermia alone did not alter tumor progression. While cytotoxic TS.mTCE αEGFRvIII CAR T cells led to an initial reduction in tumor burden, they failed to sustain long-term tumor control. By contrast, FUS treated mice receiving TS.mTCE CAR T cell cohorts exhibited decreased tumor growth, with luminescence imaging showing markedly lower signals compared to all other control groups (Fig. 1g-h). Taken together, these findings provide support that metronomic and sonothermal activation of TS.mTCE αEGFRvIII CAR T cells can overcome intratumoral immunosuppression by in situ mTCE production to reduce the population of M-MDSCs and demonstrate clinically relevant methods to remotely modulate tumor micronenvionment by CAR T cells across an intact skull.

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