CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.

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The efficacy of T-cell therapy is inhibited by various tumor-associated immunosuppressive ligands and soluble factors. Such inhibitory signals turn specific T-cell signaling pathways on or off, impeding the anticancer functions of T cells. Many studies have focused on PD-1 or CTLA-4 blockade to invigorate T-cell functions through CD28/B7 signaling, but obtaining robust clinical outcomes remains challenging. In this study, we use CRISPR/Cas9 to potentiate T-cell function by increasing CD3 signaling via knockout of diacylglycerol kinase (DGK), an enzyme that metabolizes diacylglycerol to phosphatidic acid. Knockout of DGK augmented the effector functions of CAR-T cells via increased TCR signaling. DGK knockout from CAR-T cells rendered them resistant to soluble immunosuppressive factors such as TGFβ and prostaglandin E2 and sustained effector functions under conditions of repeated tumor stimulation. Moreover, DGK knockout caused significant regression of U87MGvIII glioblastoma tumors through enhanced effector functions in a xenograft mouse model. Collectively, our study shows that knockout of DGK effectively enhances the effector functions of CAR-T cells, suggesting that CRISPR/Cas9-mediated knockout of DGK could be applicable as part of a multifaceted clinical strategy to treat solid cancers. This novel study demonstrates efficient ablation of diacylglycerol kinase in human CAR-T cells that leads to improved antitumor immunity and may have significant impact in human cancer immunotherapy. .

& Johnson, Poseida Therapeutics, and IASO Biotherapeutics; and serves on the medical advisory board and scientific advisory board (SAB) for IASO Biotherapeutics. BLL and CHJ are scientific founders of Tmunity Therapeutics, for which they have founder's stock. BLL, MMD, CHJ, and JAF have received royalties from Tmunity Therapeutics. CHJ and JAF are founders of DeCART Therapeutics. BLL is a consultant for Novartis and Terumo and SAB member for Avectas, Patheon/Thermo Fisher Scientific Viral Vector Services, Immuneel, In8bio, Ori Biotech, and Vycellix. MMD is a consultant and member of the SAB for Cellares Corporation. SFL is a consultant for Gilead/ Kite. JAF is a consultant for Guidepoint and LEK Consulting.

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1 , encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]–expressing CD8 + CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]–expressing CD8 + T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7 -dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)–driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3 + CD8 + and toward TCF1 + CD8 + to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.

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Chimeric antigen receptor (CAR) T cell therapy has come of age, offering a potentially curative option for patients who are refractory to standard anti-cancer treatments. The success of CAR T cell therapy in the setting of acute lymphoblastic leukemia and specific types of B cell lymphoma led to rapid regulatory approvals of CD19-directed CAR T cells, first in the United States and subsequently across the globe. Despite these major milestones in the field of immuno-oncology, growing experience with CAR T cells has also highlighted the major limitations of this strategy, namely challenges associated with manufacturing a bespoke patient-specific product, intrinsic immune cell defects leading to poor CAR T cell function as well as persistence, and/or tumor cell resistance resulting from loss or modulation of the targeted antigen. In addition, both onand off-tumor immunotoxicities and the financial burden inherent in conventional cellular biomanufacturing often hamper the success of CAR T cell-based treatment approaches. Herein, we provide an overview of the opportunities and challenges related to the first form of gene transfer therapy to gain commercial approval in the United States. Ongoing advances in the areas of genetic engineering, precision genome editing, toxicity mitigation methods and cell manufacturing will improve the efficacy and safety of CAR T cells for hematologic malignancies and expand the use of this novel class of therapeutics to reach solid tumors.