DNMT3A deletion preserves CAR T cell functionality during prolonged stimulation.

Developing CAR T cells for acute myeloid leukemia (AML) has been hampered by a paucity of targets that are expressed on AML blasts and not on hematopoietic progenitor cells (HPCs). Here we demonstrate that GRP78 is expressed on the cell surface of primary AML blasts but not HPCs. To target GRP78, we generate T cell expressing a GRP78-specific peptide-based CAR, which show evidence of minimal fratricide post activation/transduction and antigen-dependent T cell differentiation. GRP78-CAR T cells recognize and kill GRP78-positive AML cells without toxicity to HPCs. In vivo, GRP78-CAR T cells have significant anti-AML activity. To prevent antigen-dependent T cell differentiation, we block CAR signaling and GRP78 cell surface expression post activation by using dasatinib during GRP78-CAR T cell manufacturing. This significantly improves their effector function in vitro and in vivo. Thus, targeting cell surface GRP78-positive AML with CAR T cells is feasible, and warrants further active exploration.

Highlights d CD19-CAR T cells undergo extensive DNA methylation reprogramming during therapy d DNA methylation status of the CAR T cell product can predict in vivo expansion d CD8 + CD19-CAR T cells acquire exhaustion-associated DNA methylation programs d Antigen-positive tumor relapse does not elicit CAR T cell expansion

The inability of chimeric antigen receptor (CAR) T cells to sustain their effector function after repeated exposure to tumor cells is a major obstacle to their success in patients with solid tumors. To overcome this limitation, we designed a novel chimeric cytokine receptor to create an autocrine loop that links activation-dependent GM-CSF production by CAR T cells to IL18 receptor signaling (GM18). Expression of GM18 in CAR T cells enhanced their effector function in an antigen- and activation-dependent manner. In repeat stimulation assays, which mimic chronic antigen exposure, CAR.GM18 T cells had a significantly greater ability to expand and produce cytokines in comparison with their unmodified counterparts targeting EPHA2 or HER2. In vivo, CAR.GM18 T cells induced tumor regression at cell doses at which standard CAR T cells were ineffective in two solid tumor xenograft models. Thus, our study highlights the potential of hijacking cytokines that are physiologically secreted by T cells to bolster their antitumor activity. Significance: We designed a chimeric cytokine receptor (GM18) that links CAR T-cell activation to MYD88 signaling. GM18 endows CAR T cells with sustained effector function in the setting of chronic antigen exposure, resulting in potent antitumor activity in preclinical solid tumor models. This article is highlighted in the In This Issue feature, p. 1601

Current chimeric antigen receptor-modified (CAR) T cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T cell receptor (TCR) sequencing dataset using pre- and post-infusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL). We identified cytotoxic post-infusion cells with identical TCRs to a subset of pre-infusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared to other pre-infusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor, TOX. Collectively, these results demonstrate diverse effector potentials within pre-infusion CAR T cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytical framework for elucidating the mechanisms underlying effector development in CAR T cell products.