DISRUPTION OF CTLA-4 BUT NOT PD-1 ENHANCES AND INVIGORATES CHIMERIC ANTIGEN RECEPTOR T CELLS

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has achieved remarkable success in treating B-cell malignancies, however, some patients fail to respond due to poor fitness of the adoptively transferred T cells. To potentially increase the number of patients that could benefit from CART19 therapy, we investigated whether CRISPR-Cas9-mediated disruption of PDCD1 and/or CTLA- 4 in patient T cells could enhance efficacy and restore CART cell function. Indeed, we demonstrate, in both an in vitro model of CART cell dysfunction and xenograft mouse models, that disruption of CTLA-4, but not PDCD1 or PDCD1 and CTLA-4 results in superior CART cell performance evidenced by increased proliferation, cytokine production, and superior anti-tumor response. Furthermore, CART19 cells manufactured from T cells lacking CTLA-4 maintain CAR expression on the T cell surface under conditions of chronic antigen exposure, which is a driver of CAR- dysfunction. To understand mechanistically how CTLA-4 disruption contributes to the increased anti-tumor efficacy of CART19 cells we perform transcriptomic analyses and identify upregulated pathways, aligning with the observed therapeutic superiority, including granzyme A and B signaling, cell cycle regulation, and cytokine signaling. Moreover, CTLA-4 disrupted CART19 cells display unopposed CD28 signaling, which is observed by pathway analyses. Importantly we confirm the clinical relevance of these results, establishing that depletion of CTLA-4 rescues the function of T cells from leukemia patients that previously failed CART cell treatment and enables the manufacture of potent CART19 products from CLL patient T cells that previously produced an ineffective product. Our findings reveal that disruption of CTLA-4 invigorates dysfunctional patient T cells, providing a strategy for increasing the number of patients that respond to CART19 therapy.