IL15 modification enables CAR T cells to act as a dual targeting agent against tumor cells and myeloid-derived suppressor cells in GBM.

INTRODUCTION: The immunosuppressive tumor microenvironment (TME) is a major barrier to the efficacy of chimeric antigen receptor T cells (CAR-T cells) in glioblastoma (GBM). Transgenic expression of IL15 is one attractive strategy to modulate the TME. However, at present, it is unclear if IL15 could be used to directly target myeloid-derived suppressor cells (MDSCs), a major cellular component of the GBM TME. Here, we explored if MDSC express IL15Rα and the feasibility of exploiting its expression as an immunotherapeutic target. METHODS: RNA-seq, RT-qPCR, and flow cytometry were used to determine IL15Rα expression in paired peripheral and tumor-infiltrating immune cells of GBM patients and two syngeneic murine GBM models. We generated murine T cells expressing IL13Rα2-CARs and secretory IL15 (CAR.IL15s) or IL13Rα2-CARs in which IL15 was fused to the CAR to serve as an IL15Rα-targeting moiety (CAR.IL15f), and characterized their effector function in vitro and in syngeneic IL13Rα2+glioma models. RESULTS: IL15Rα was preferentially expressed in myeloid, B, and dendritic cells in patients' and syngeneic GBMs. In vitro, CAR.IL15s and CAR.IL15f T cells depleted MDSC and decreased their secretion of immunosuppressive molecules with CAR.IL15f T cells being more efficacious. Similarly, CAR.IL15f T cells significantly improved the survival of mice in two GBM models. TME analysis showed that treatment with CAR.IL15f T cells resulted in higher frequencies of CD8+T cells, NK, and B cells, but a decrease in CD11b+cells in tumors compared with therapy with CAR T cells. CONCLUSIONS: We demonstrate that MDSC of the glioma TME express IL15Ra and that these cells can be targeted with secretory IL15 or an IL15Rα-targeting moiety incorporated into the CAR. Thus, IL15-modified CAR T cells act as a dual targeting agent against tumor cells and MDSC in GBM, warranting their future evaluation in early-phase clinical studies.

Indocarbocyanine nanoparticles extravasate and distribute better than liposomes in brain tumors.

Glioblastoma (GBM) is the most devastating and aggressive brain tumor in adults. Hidden behind the blood-brain and blood-tumor barriers (BBTB), this invasive type of brain tumor is not readily accessible to nano-sized particles. Here we demonstrate that fluorescent indocarbocyanine lipids (ICLs: DiD, DiI) formulated in PEGylated lipid nanoparticle (PLN) exhibit highly efficient penetration and accumulation in GBM. PLN-formulated ICLs demonstrated more efficient penetration in GBM spheroids and organoids in vitro than liposomal ICLs. Over 82% of the tumor's extravascular area was positive for ICL fluorescence in the PLN group versus 13% in the liposomal group just one hour post-systemic injection in the intracranial GBM model. Forty-eight hours post-injection, PLN-formulated ICLs accumulated in 95% of tumor myeloid-derived suppressor cells and macrophages, 70% of tumor regulatory T cells, 50% of tumor-associated microglia, and 65% of non-immune cells. PLN-formulated ICLs extravasated better than PEGylated liposomal doxorubicin and fluorescent dextran and efficiently accumulated in invasive tumor margins and brain-invading cells. While liposomes were stable in serum in vitro and in vivo, PLNs disassembled before entering tumors, which could explain the differences in their extravasation efficiency. These findings offer an opportunity to improve therapeutic cargo delivery to invasive GBM.