A Costimulatory CAR Improves TCR-based Cancer Immunotherapy.

T-cell receptors (TCR) recognize intracellular and extracellular cancer antigens, allowing T cells to target many tumor antigens. To sustain proliferation and persistence, T cells require not only signaling through the TCR (signal 1), but also costimulatory (signal 2) and cytokine (signal 3) signaling. Because most cancer cells lack costimulatory molecules, TCR engagement at the tumor site results in incomplete T-cell activation and transient antitumor effects. To overcome this lack of signal 2, we genetically modified tumor-specific T cells with a costimulatory chimeric antigen receptor (CoCAR). Like classical CARs, CoCARs combine the antigen-binding domain of an antibody with costimulatory endodomains to trigger T-cell proliferation, but CoCARs lack the cytotoxic CD3ζ chain to avoid toxicity to normal tissues. We first tested a CD19-targeting CoCAR in combination with an HLA-A*02:01-restricted, survivin-specific transgenic TCR (sTCR) in serial cocultures with leukemia cells coexpressing the cognate peptide-HLA complex (signal 1) and CD19 (signal 2). The CoCAR enabled sTCR+ T cells to kill tumors over a median of four additional tumor challenges. CoCAR activity depended on CD19 but was maintained in tumors with heterogeneous CD19 expression. In a murine tumor model, sTCR+CoCAR+ T cells improved tumor control and prolonged survival compared with sTCR+ T cells. We further evaluated the CoCAR in Epstein-Barr virus-specific T cells (EBVST). CoCAR-expressing EBVSTs expanded more rapidly than nontransduced EBVSTs and delayed tumor progression in an EBV+ murine lymphoma model. Overall, we demonstrated that the CoCAR can increase the activity of T cells expressing both native and transgenic TCRs and enhance antitumor responses.

Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies and is now being developed for solid tumors and chronic viral infections. Although clinical trials have demonstrated the curative potential of CAR T cell therapy, a substantial and well-established limitation is the heightened contraction and transient persistence of CAR T cells during prolonged antigen exposure. The underlying mechanism(s) for this dysfunctional state, often termed CAR T cell exhaustion, remains poorly defined. Here, we report that exhaustion of human CAR T cells occurs through an epigenetic repression of the T cell's multipotent developmental potential. Deletion of the de novo DNA methyltransferase 3 alpha (DNMT3A) in T cells expressing first- or second-generation CARs universally preserved the cells' ability to proliferate and mount an antitumor response during prolonged tumor exposure. The increased functionality of the exhaustion-resistant DNMT3A knockout CAR T cells was coupled to an up-regulation of interleukin-10, and genome-wide DNA methylation profiling defined an atlas of genes targeted for epigenetic silencing. This atlas provides a molecular definition of CAR T cell exhaustion, which includes many transcriptional regulators that limit the "stemness" of immune cells, including CD28, CCR7, TCF7, and LEF1. Last, we demonstrate that this epigenetically regulated multipotency program is firmly coupled to the clinical outcome of prior CAR T cell therapies. These data document the critical role epigenetic mechanisms play in limiting the fate potential of human T cells and provide a road map for leveraging this information for improving CAR T cell efficacy.