title: Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.
abstract: Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection. The most successful CARs used to date are those targeting CD19 (ref. 2), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies. CARs are typically transduced into the T cells of a patient using γ-retroviral vectors or other randomly integrating vectors, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific interventions in human cells, including targeted gene delivery to the CCR5 and AAVS1 loci. Here we show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.
authors.affiliation: Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, New York 10065, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Eyquem
authors.affiliation: Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, New York 10065, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Mansilla-Soto
authors.affiliation: Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, New York 10065, USA.
authors.forename: Theodoros
authors.initials: T
authors.lastname: Giavridis
authors.affiliation: Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, New York 10065, USA.
authors.forename: Sjoukje J C
authors.initials: SJ
authors.lastname: van der Stegen
authors.affiliation: Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, New York 10065, USA.
authors.forename: Mohamad
authors.initials: M
authors.lastname: Hamieh
authors.affiliation: Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Cunanan
authors.affiliation: Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, New York 10065, USA.
authors.forename: Ashlesha
authors.initials: A
authors.lastname: Odak
authors.affiliation: Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.
authors.forename: Mithat
authors.initials: M
authors.lastname: Gönen
authors.affiliation: Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, New York 10065, USA.
authors.forename: Michel
authors.initials: M
authors.lastname: Sadelain
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000276:immunology; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D004195:Disease Models, Animal; D000072669:Gene Editing; D005786:Gene Expression Regulation; D056426:Genetic Loci / Q000235:genetics; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000276:immunology* / Q000628:therapy; D011401:Promoter Regions, Genetic / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D057170:Translational Research, Biomedical
chemical_list: D018941:Antigens, CD19; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta