Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8⁺ memory T cell progenitors that can become either functional stem-like T (T_STEM) cells or dysfunctional T progenitor exhausted (T_PEX) cells. To that end, we demonstrated that T_STEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate T_STEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, T_STEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4⁺ T cells during T_STEM-like CAR-T cell production. Adoptive transfer of T_STEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of T_STEM-like CAR-T cells and an increased memory T cell pool. Last, T_STEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8⁺CAR⁺ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated T_STEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

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