High-avidity Cathepsin G specific CAR-T cells for the treatment of Acute Myeloid Leukemia

Chimeric antigen receptor (CAR) T cells specific for myeloid-associated antigens expressed on the cell surface of acute myeloid leukemia (AML) cause depletion of normal myeloid progenitor cells. We developed a CAR specific for a human Leucocyte Histocompatibility Antigen (HLA)-A*02:01-restricted peptide of the cathepsin G protein, which is a myeloid-restricted protein expressed in the cytoplasm of myeloid leukemic blasts. Cathepsin G-specific CAR (CG1.CAR) T cells were further engineered to increase their functional avidity. Specifically, we developed CG1.CAR-T cells co-expressing the lymphocyte-specific protein tyrosine kinase (LCK) and duplicated CD3ζ chain, which allows the functional recognition of the CG1 peptide as low as 0.025 µM. Optimized CG1.CAR T cells displayed antileukemia effects in vitro and in vivo in AML patientderived-xenotransplant (PDX) mouse models and did not cause hematopoietic toxicity in colony assays and humanized mice. Mechanistically, LCK overexpression in CG1.CAR-T cells caused transcriptional modifications characterized by the overexpression of mitochondrial-encoded electron transport chain components that were correlated with increased mitochondrial mass and improved respiratory capacity. Based on these data, CG1.CAR-T cells hold clinical potential for the treatment of AML. To investigate the effect of LCK overexpression on CAR signaling, we performed transcriptomic profiling of CAR-T cells following antigen stimulation. Human PBMCs were transduced with either CAR.CG1 or CAR.CG1.ζζ.LCK constructs and expanded for 10 days in the presence of IL-7 and IL-15. On day 10, cells were rested for 24 hours in cytokine-free medium. On day 11, CAR-T cells were co-cultured with U937/HLA-A2 tumor cells at an effector-to-target (E:T) ratio of 1:1 (0.5 × 10⁶ cells per condition). Co-culture was maintained for either 24 hours or 5 days. CAR-T cells were sorted by flow cytometry based on expression of the HA tag incorporated in the CAR constructs. RNA was extracted for sequencing at three time points: (1) prior to antigen stimulation (baseline), (2) 24 hours post-stimulation, and (3) 5 days post-stimulation. Samples from both CAR.CG1 and CAR.CG1.ζζ.LCK groups were included at all time points. *************************************************************** Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access. ***************************************************************