277 SynNotch-CAR T cells demonstrate potent anti-tumor efficacy in a preclinical immunocompetent mouse model for glioblastoma

Background

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor. Recent immunotherapy studies, including CAR T cells, have shown limited success in GBM due to multiple barriers, such as on-target off-tumor toxicity, the blood-brain barrier, and the immunosuppressive tumor microenvironment (TME). To overcome these challenges, we recently adopted a novel synthetic Notch ‘synNotch’ receptor system and developed innovative T cell (‘synNotch-CART’) circuits based on the ‘prime-and-kill’ strategy.¹ Here, we investigated the GBM-homing and efficacy of SynNotch-CAR T cells using a preclinical immunocompetent mouse model, which, similar to GBM patients, is poorly immunogenic and unresponsive to immune checkpoint blockade.²

Methods

Epidermal growth factor receptor (EGFR) amplification and overexpression occur in approximately 40–60% of GBM patients.³ Therefore, we first expressed the extracellular domain of mouse EGFR in a syngeneic glioma cell line, SB28 (SB28-mEGFR). Next, we developed a novel synNotch-CAR (mBSYNC) circuit in which the brain-specific antigen, brevican (BCAN) primes the T cells to induce the expression of a CAR that recognizes and kills EGFR-positive cells (mBSYNC synNotch-CAR T cells). 8-week-old C57BL/6 mice bearing the SB28-mEGFR cells in the frontal lobe received a single intravenous infusion of mBSYNC synNotch-CAR or conventional CAR T cells with constitutive CAR expression on day 8 following the tumor inoculation. Trafficking of CAR T cells and host immune cells into the brains was evaluated using high-dimensional flow cytometry.

Results

Constitutive a-EGFR CAR and mBSYNC synNotch-CAR T cells mediated antigen-specific cytotoxicity against SB28-mEGFR cells in vitro. In an in vivo prospective study, on day 12 after T cell infusion, mBSYNC synNotch-CAR T cells demonstrated a higher persistence, increased infiltration of endogenous CD86⁺ and MHC-II^hi macrophages and decreased infiltration of CD206⁺ macrophages into the GBM TME compared to either untransduced or constitutive a-EGFR CAR T-cells. Lastly, a single intravenous infusion of mBSYNC synNotch-CAR T cells significantly (p < 0.01) prolonged the survival and completely eradicated the aggressive tumor in 3 of 10 mice bearing SB28-mEGFR gliomas. Conclusions mBSYNC synNotch-CAR T cells, but not conventional CAR T cells, effectively home into the brain TME, persist, and eliminate the tumor in an immunocompetent setting. The induced pro-inflammatory activity of endogenous myeloid cells could contribute to the anti-tumor efficacy mediated by synNotch-CAR T cells. Ongoing studies are aimed at the thorough characterization of tumor cells, host immune cells, and synNotch-CAR T cells in the brain TME.

References

Choe JH, Watchmaker PB, Simic MS, Gilbert RD, Li AW, Krasnow NA, Downey KM, Yu W, Carrera DA, Celli A, et al. SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma. Sci Transl Med 2021;13. 10.1126/scitranslmed.abe7378. Genoud V, Marinari E, Nikolaev SI, Castle JC, Bukur V, Dietrich PY, Okada H, Walker PR. Responsiveness to anti-PD-1 and anti-CTLA-4 immune checkpoint blockade in SB28 and GL261 mouse glioma models. Oncoimmunology 2018;7:e1501137. 10.1080/2162402X.2018.1501137. Brennan CW, Verhaak RG, McKenna A, Campos B, Noushmehr H, Salama SR, Zheng S, Chakravarty D, Sanborn JZ, Berman SH, et al. The somatic genomic landscape of glioblastoma. Cell 2013;155:462–477. 10.1016/j.cell.2013.09.034.