Image-Guided Sonogenetic Control of CAR T Cells to Produce NKG2D Bispecific T Cell Engagers Overcomes Brain Tumor Immunosuppression

Introduction: Glioblastoma (GBM) is an aggressive and highly lethal brain tumor with a median survival of less than 12 months. One key challenge in treating GBM is overcoming the profound immunosuppression within the tumor microenvironment, largely mediated by myeloid-derived suppressor cells (MDSCs). These MDSCs inhibit T cell function and promote tumor growth, posing a significant roadblock in translating the success of CAR T cell therapy for applications in GBM. 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. 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 BTEs. To locally deliver and restrict activity of immunostimulatory molecules to sites of disease, we engineer CAR T cells with thermal-sensitive gene switches to locally produce BTEs upon mild hyperthermia mediated by non-invasive, MR-guided focused ultrasound (MRgFUS) and demonstrate reduction in immunosuppressive MDSCs, thereby reducing immunosuppression and ultimately enhancing anti-tumor immunity.

Materials and

Methods: We engineered thermal gene switches with low activity at basal conditions (37°C) and high fold induction (>200-fold) following exposure to mild hyperthermia (40-42°C). Primary human T cells were transduced with thermally induced Fluc or NKG2DL-targeting BTE (TS.Fluc and TS.BTE respectively) upstream of a constitutive control CAR vector to kill intratumoral MDSCs following MR image guided hyperthermia (Fig 1a). To evaluate the effect of BTE on MDSC suppression, producer cells (mBTE.293T) were generated by transducing 293T cells with mBTE lentivirus. Bone marrow from C57BL/6J mice were flushed and cultured with mGM-CSF, IFNγ, and LPS to generate MDSCs. Murine T cells were isolated from C57BL/6J spleens, stained with cell trace violet (CTV), and activated with CD3/CD28 dynabeads. The mBTE.293T cells were co-cultured with MDSCs and activated murine T cells at various MDSC:T cell ratios (2:1, 1:1, 0.5:1, 0.25:1, 0) for 4 days. Activation (CD25 expression) and proliferation (CTV) of T cells were assessed by flow cytometry (Fig. 1b-d). Next, to evaluate the effect of local BTE production on MDSC in vivo, C57BL/6J mice were inoculated with 2×103 SB28 cells intracranially to establish GBM. 8 days following inoculation, mice received an adoptive transfer of 2.5×104 TS-mBTE CAR T cells (Fig 2e). Mild hyperthermia (41°C) was applied using MRgFUS for 10 minutes on day 9 to locally activate the thermal gene switches and produce BTEs. On day 11, mice were sacrificed, brains dissociated, and flow analysis was performed to quantify CAR+ T cells and MDSCs (Fig 2f).

Results, Conclusions, and Discussions: We postulated that NKG2DL targeting BTEs will engage T cells and redirect their cytotoxicity towards MDSCs and therefore recover T cell function in an immunosuppressive tumor microenvironment (Fig 1a). We first evaluated the functional effect of BTEs by coincubating T cells with MDSCs at different E:T ratios in mBTE.293T or WT.293T conditioned media. We observed significant T cell activation and proliferation, indicated by increased CD25 expression and CTV dilution (Fig 1b). Quantification of CD25 mean fluorescent intensity (MFI) and CTV mediated expansion demonstrated increased T cell activation and expansion in mBTE.293T conditioned media, especially prominent at MDSC: T cell ratios between 1:1 and 0.25:1 (Fig. 1c). We next evaluated the in vivo efficacy of this approach in an immunocompetent GBM murine model. Following ACT of TS.mBTE CAR T cells and MRgFUS-mediated hyperthermia (Fig. 1e), we observed a significant reduction in MDSC populations within the tumor microenvironment when compared to unheated controls (Fig. 1f).
Taken together, our studies demonstrate that BTEs can suppress T cell activation and proliferation and are heavily recruited to GBM tumors – a technical challenge that limits effective translation of immunotherapies. Our results provide evidence that the thermally controlled local production of BTEs by engineered CAR T cells can effectively reduce immunosuppression and has the potential to enhance anti-tumor immunity in glioblastoma. This approach leverages the precision of MRgFUS to produce immunostimulatory molecules at the tumor site, offering a promising strategy to improve the efficacy of CAR T cell therapies in solid brain tumors.

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