Immune Synapse Optimized GRP78-Specific CAR T Cells Elicit an Improved Antitumor Response Against DIPGs

Abstract Diffuse intrinsic pontine gliomas (DIPGs) are highly lethal pediatric brain tumors and a leading cause of cancer-related death in children. There is currently no cure for DIPG, highlighting an urgent need for novel therapeutics. Chimeric antigen receptor (CAR)-engineered T-cell therapy offers great promise for DIPG treatment; however, several limitations need to be addressed. In this study, we targeted a novel tumor associated antigen GRP78 after showing it is reliably expressed on the cell surface of DIPGs. To test the anti-DIPG activity of GRP78-CAR T cells, we evaluated cytotoxicity, persistence, and cytokine secretion in vitro. We found that GRP78-CAR T cells kill DIPGs but fail to proliferate and secrete cytokines compared to positive control, U87. In vivo studies further confirmed that GRP78-CAR T cells eradicated U87 tumors but not DIPGs. Based on these findings, we concluded that DIPGs cannot trigger efficient CAR T cell signaling. Because CAR T cell effector function is influenced by antigen density, we quantified the relative GRP78 expression on DIPGs and other brain tumors, including U87. We found that GRP78 is comparatively lowly expressed on DIPGs. Live-cell imaging of calcium flux in CAR T cells further revealed that T cell activation is lower upon interaction with DIPGs than U87s, suggesting that CAR-T cells form incomplete or dysfunctional immune synapses (IS) against DIPGs. To improve IS function, we knocked-out RASA2, a TCR downstream signaling inhibitor. Our results show that T cells lacking RASA2 have an improved IS reflected by bigger synaptic areas, increased lytic granules, and pZAP70 accumulation at the synapse. RASA2 KO further increased calcium influx and proliferation capacity of GRP78-CAR T cells against DIPGs. Thus, this study confirms that antigen density controls CAR T cell antitumor activity, and improving the IS formation of CAR T cells elicits enhanced antitumor response against low antigen-expressing tumor cells.