PI3Kδ/γ inhibition promotes CART cell epigenetic and metabolic re-programming to enhance anti-tumor cytotoxicity

Abstract Current limitations using chimeric antigen receptor (CART) cells to treat patients with hematological cancers include limited expansion and persistence in vivo that contribute to cancer relapse. Patients with chronic lymphocytic leukemia (CLL) have terminally differentiated T-cells with an exhausted phenotype and experience low complete response rates following autologous CART therapy. Since PI3K inhibitor therapy is associated with the development of T-cell mediated autoimmunity, we studied the effects of inhibiting PI3Kδ and γ isoforms during manufacturing of CART cells prepared from CLL patients. Dual PI3Kδ/γ inhibition normalized CD4:CD8 ratios, maximized numbers of CD8+ T-stem cell memory (Tscm), naive, and central memory T-cells with dose-dependent decreases in expression of the TIM-3 exhaustion marker. CART cells manufactured with duvelisib (Duv-CART cells) showed significantly increased in vitro cytotoxicity against CD19+ CLL targets due to increased frequencies of CD8+ CART. Duv-CART had increased expression of mitochondrial fusion gene MFN2, with an associated increase in the relative content of mitochondria. Duv-CART exhibit increased SIRT1 and TCF1/7 expression, which correlated with epigenetic reprograming of Duv-CART T-cell toward stem-like properties. Following transfer to NOG mice engrafted with a human CLL cell line, Duv-CART cells expressing either a CD28 or 41BB costimulatory domain demonstrated significantly increased in vivo expansion of CD8+ CART, faster elimination of CLL, and longer persistence; Duv-CART cells significantly enhanced survival of CLL-bearing mice compared with conventionally manufactured CART. In conclusion, exposure of CART to a PI3Kδ/γ inhibitor during manufacturing enriched the CART product for CD8+ CART cells with stem-like qualities and enhanced efficacy in eliminating CLL in vivo.