Immunometabolic determinants of long-term response in leukemia patients receiving CD19 CAR T cell therapy

Although most patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) receiving CD19-targeted chimeric antigen receptor (CAR) T cell therapy achieve remission, loss of CAR T cell functionality and subsequent relapse remains an unmet therapeutic need. We applied an integrative approach to study the immunometabolism of pre- and post-infusion CD19-CAR T cells of patients with relapsed/refractory B-ALL. Pre-infusion CAR T cells of long-term responders (LTR) had increased oxidative phosphorylation, fatty acid oxidation, and pentose phosphate pathway activities, higher mitochondrial mass, tighter cristae, and lower mTOR expression compared to products of short-term responders. Post-infusion CAR T cells in bone marrow (BM) of LTR had high immunometabolic plasticity and mTOR-pS6 expression supported by the BM microenvironment. Transient inhibition of mTOR during manufacture induced metabolic reprogramming and enhanced anti-tumor activity of CAR T cells. Our findings provide insight into immunometabolic determinants of long-term response and suggest a therapeutic strategy to improve long-term remission. Overall design: Generation of CAR T cells with rapamycin. Enriched Tn/mem cells from leukapheresis products were stimulated with GMP Human T-expander CD3/CD28 Dynabeads™ (Dynal Biotech Cat#11141D) at a ratio of 1:3 (T cell:bead) overnight. Activated T cells were subsequently transduced with CD19R(EQ):CD28:?/EGFRt clinical construct at MOI=1 in RPMI 1640 containing 10% FBS and 5µg/ml protamine sulfate (Fresenius Kabi, 22905), 50U/mL rhIL-2 (Novartis Pharmaceuticals, NDC0078-0495-61), and 0.5 ng/mL rhIL-15 (CellGenix, 1013-050). Beginning 4 days after bead stimulation, CAR T cells were treated with rapamycin (50 nmol/L) (LC Laboratories, R-5000) or vehicle (DMSO) and refreshed with media containing rapamycin or vehicle every other day. Six days after stimulation, CD3/CD28 Dynabeads™ beads were removed using a DynaMag™-5 Magnet (Invitrogen, 12303D), and cells were maintained at a concentration of 1x106 cells/mL in RPMI 1640/10% FBS, 25U/mL rhIL-2, and 0.25 ng/mL rhIL-15 throughout T cell expansion.