CRISPR-engineered T cells in patients with refractory cancer

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Abstract

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 I 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 PD-1 ( PDCD1), was performed to improve anti-tumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Though 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.

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Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.

Leonid Cherkassky, Aurore Morello, Jonathan Villena-Vargas … (2016)

Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1-mediated (PD-1-mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB-based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies.

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A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: long-term follow-up and correlates with response.

Paul Robbins, Sadik Kassim, Thai Tran … (2015)

Although adoptive cell therapy can be highly effective for the treatment of patients with melanoma, the application of this approach to the treatment of other solid tumors has been limited. The observation that the cancer germline (CG) antigen NY-ESO-1 is expressed in 70% to 80% and in approximately 25% of patients with synovial cell sarcoma and melanoma, respectively, prompted us to perform this first-in-man clinical trial using the adoptive transfer of autologous peripheral blood mononuclear cells that were retrovirally transduced with an NY-ESO-1-reactive T-cell receptor (TCR) to heavily pretreated patients bearing these metastatic cancers.

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A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells

Christopher Vakulskas, Daniel Dever, Garrett R Rettig … (2018)

Translation of the CRISPR/Cas9 system to human therapeutics holds high promise. Specificity remains a concern, however, especially when modifying stem cell populations. We show that existing rationally-engineered Cas9 high fidelity variants have reduced on-target activity using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells. We also show that the HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Val mutation in SCD patient derived HSPCs. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome editing applications.