Remote activation of CAR T cells by MR-guided focused ultrasound for treatment of brain tumors

Introduction:: Breast cancer is the most diagnosed cancer in women resulting in 500,000 deaths annually worldwide. Moreover, metastasis of disease in the brain or central nervous system is observed in up to 50% of HER2+ breast cancer patients resulting in poor survival rates of less than 2 years. Treating patients with breast cancer brain metastasis (BCBM) remains an unmet clinical need as challenges with disease management are multifaceted. Systemic administration of immunomodulatory biologics such as monoclonal antibodies and bispecific T cell engagers (BTEs) are confronted with limited efficacy due to their dose limiting toxicities and inability to adequately cross the blood-brain barrier (BBB). Although small molecule inhibitors of HER2 are capable of crossing the BBB and are clinically approved, their efficacy is limited in brain metastasis with heterogenous HER2 expression and can lead to tumor antigen escape and disease relapse. 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 enhanced therapeutic efficacy in mitigating outgrowth of a heterogenous brain tumor model of antigen escape.

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 αHER2 CAR vector (Fig. 1a top). MDA-MB-468 tumor cells with heterogenous expression of HER2 (3:1 ratio of HER2+ to HER2-) were inoculated in the brain of NSG mice as a preclinical model of BCBM. one day after adoptive transfer of 5×10⁵ engineered CARs, 10 minutes of pulsed heating (Fig. 1a bottom) was deposited remotely using MRgFUS to locally produce BTEs and redirect CARs towards HER2- tumor cells (Fig. 1b). in vivo activation of TS.Fluc αHER2 CAR T was quantified by IVIS 10 hours post heating. Afterwards, untransduced cells or TS.BTE αHER2 CAR T cells were transferred and therapeutic efficacy evaluated by quantifying tumor burden of Fluc+ HER2- tumor cells. Heated cohorts received two thermal treatments three days apart.

Results, Conclusions, and Discussions:: Remote activation of TS.Fluc αHER2 CAR T cells by MRgFUS resulted in a ~10 fold increase in luminescent signal indicating transgene activation in the brain of tumor bearing mice (Fig. 1c). Moreover, after adoptive transfer of untransduced and TS.BTE αHER2 CAR T cells, we observed a significant tumor regression of HER2- tumor cells in CAR treated mice when compared to cohorts which received untransduced T cells (Fig. 1d). However, cohorts which received CARs and remained unheated, quickly relapsed 8 days post initial treatment while mice which received both CARs and heat, had significantly lower tumor burden for over two weeks after initial treatment and displayed about a 100-fold reduction in tumor luminescence when compared to control cohorts.

The ability to control T cells as drug delivery vehicles has the potential to improve therapy in heterogenous brain tumors. Here, we demonstrate that local, thermally induced production of NKG2DL BiTEs by MRgFUS can enhance therapy for BCBM. Looking forward, our strategy complements existing clinical protocols to enhance T cell infiltration into brain tumors following systemic administration to locally deliver immunomodulatory therapeutics remotely.

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