(J-384) Developing 4th Generation CAR T Cell microRNA Biofactories for Solid Tumor Treatment

Translation of CAR T cell therapy to solid tumors must overcome many challenges including tumor infiltration, immunosuppressive tumor microenvironments, and antigen escape. We have developed 4^th generation-like CAR-T-miR cells which use co-stimulatory domains to produce and export microRNA when stimulated by the targeted cancer antigen IL13Rα2 (Figure 1A). These CAR-T-miR cell microRNA biofactories utilize the ability of microRNA, specifically miR-34a-5p, to simultaneously regulate multiple genetic pathways and enhance the ability of CAR T cells for solid tumor treatment. In this study, we explore the effects of CAR-T-miR cells on glioblastoma multiforme (GBM), a highly aggressive tumor with an average five-year survival rate of less than 5%.

The CAR construct was cloned with a modified interleukin 13 (IL13 E12Y) binding domain, which has exhibited high affinity to IL13Rα2 in GBM cells, and a pre-miR34a sequence under the NFAT-IL2 minimal promoter (Figure 1D). Lentiviral transfection was used to deliver the plasmid to Jurkat and T cells. CAR Jurkat cells were the proof of concept for CAR T cells. Flow cytometry was used to measure CAR expression after transfection and puromycin selection. Exosome from IL13Rα2 stimulated and non-stimulated CAR Jurkat cells was isolated to show the production and exportation of miR-34a (Figure 1B). U87 GBM cells were co-cultured with CAR cells for 48 hours. Survival of U87 cells stained with Cell Tracker Green CMFDA dye was measured via flow cytometry and SpectraMax iD3 microplate reader then normalized to the naïve treated group (Figure 1C).

The CAR-miR Jurkat cells successfully produced and exported miR-34a-5p via their exosomes after stimulation of the CAR molecule (Figure 1B). CAR-miR Jurkat and T cells both exhibited improved cytotoxicity against GBM cells in vitro as seen through the decreased survival of the U87 cells (Figure 1C).

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Figure 1. A. Graphical representation of the CAR-T-miR cell, microRNA biofactories with production and exportation of microRNA triggered by the CAR T cell binding to the antigen. B. Histograms reporting the mean value ± SD of fold change for CAR Jurkat exportation of miR-34a-5p in exosomes with and without stimulation of the CAR construct by IL13R α2 beads (n = 2 per group). C. Histograms reporting the percent survival ± SD of U87 GBM cells after 48-hour co-culture with either naive (non-transfected), CAR, or CAR-miR cells (n = 2 per group). Significant difference of * p < 0.05, ** p < 0.01. D. Graphical representation of the 4^th generation CAR-miR cell design including the primary CAR construct and pre-miR sequence under the NFAT-IL2 promoter.

CAR-miR cells successfully exported more miR-34a-5p and exhibited higher cytotoxicity than CAR cells. Ongoing studies include continued in vitro testing with CAR-T-miR cells, testing of CAR-T-miR cells in other solid tumors such as metastatic melanoma, and in vivo studies.