Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection1. 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 malignancies3. CARs are typically transduced into the T cells of a patient using γ-retroviral4 vectors or other randomly integrating vectors5, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing6,7,8. Recent advances in genome editing enable efficient sequence-specific interventions in human cells9,10, including targeted gene delivery to the CCR5 and AAVS1 loci11,12. 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.
This is a preview of subscription content
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Jensen, M. C. & Riddell, S. R. Designing chimeric antigen receptors to effectively and safely target tumors. Curr. Opin. Immunol . 33, 9–15 (2015)
Brentjens, R. J. et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nat. Med . 9, 279–286 (2003)
Sadelain, M. CAR therapy: the CD19 paradigm. J. Clin. Invest . 125, 3392–3400 (2015)
Sadelain, M. & Mulligan, R. C. Efficient retroviral-mediated gene transfer into murine primary lymphocytes. Ninth International Immunology Congress, Budapest. 88:34. (1992)
Wang, X. & Rivière, I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol. Ther. Oncolytics . 3, 16015 (2016)
Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther . 16, 1241–1246 (2005)
Rivière, I., Dunbar, C. E. & Sadelain, M. Hematopoietic stem cell engineering at a crossroads. Blood 119, 1107–1116 (2012)
von Kalle, C., Deichmann, A. & Schmidt, M. Vector integration and tumorigenesis. Hum. Gene Ther . 25, 475–481 (2014)
Wright, A. V., Nuñez, J. K. & Doudna, J. A. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164, 29–44 (2016)
Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat. Rev. Genet . 17, 300–312 (2016)
Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat. Methods 8, 861–869 (2011)
Sather, B. D. et al. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci. Transl. Med. 7, 307ra156 (2015)
Brentjens, R. J. et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 5, 177ra38 (2013)
Wang, J. et al. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res . 44, e30 (2016)
Hubbard, N. et al. Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome. Blood 127, 2513–2522 (2016)
Corthay, A., Nandakumar, K. S. & Holmdahl, R. Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha-chains and of their potential role in autoimmunity. J. Autoimmun . 16, 423–429 (2001)
de Vree, P. J. et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat. Biotechnol . 32, 1019–1025 (2014)
Zhao, Z. et al. Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells. Cancer Cell 28, 415–428 (2015)
Blackburn, S. D. et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat. Immunol . 10, 29–37 (2009)
Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med . 17, 1290–1297 (2011)
Gallardo, H. F., Tan, C. & Sadelain, M. The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes. Gene Ther . 4, 1115–1119 (1997)
Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med . 21, 581–590 (2015)
Sommermeyer, D. et al. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30, 492–500 (2016)
Frigault, M. J. et al. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer Immunol. Res . 3, 356–367 (2015)
Schrum, A. G., Turka, L. A. & Palmer, E. Surface T-cell antigen receptor expression and availability for long-term antigenic signaling. Immunol. Rev . 196, 7–24 (2003)
Liu, H., Rhodes, M., Wiest, D. L. & Vignali, D. A. On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 13, 665–675 (2000)
Call, M. E. & Wucherpfennig, K. W. The T cell receptor: critical role of the membrane environment in receptor assembly and function. Annu. Rev. Immunol . 23, 101–125 (2005)
Allison, K. A. et al. Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells. eLife 5, e10134 (2016)
Schietinger, A. & Greenberg, P. D. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends Immunol . 35, 51–60 (2014)
Wherry, E. J. & Kurachi, M. Molecular and cellular insights into T cell exhaustion. Nat. Rev. Immunol . 15, 486–499 (2015)
Rivière, I., Brose, K. & Mulligan, R. C. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc. Natl Acad. Sci. USA 92, 6733–6737 (1995)
Maher, J., Brentjens, R. J., Gunset, G., Rivière, I. & Sadelain, M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ/CD28 receptor. Nat. Biotechnol . 20, 70–75 (2002)
Gong, M. C. et al. Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen. Neoplasia 1, 123–127 (1999)
Gade, T. P. et al. Targeted elimination of prostate cancer by genetically directed human T lymphocytes. Cancer Res . 65, 9080–9088 (2005)
We thank I. Rivière (MSKCC) for helpful discussion and for reviewing the manuscript. We thank the SKI Flow Cytometry core facility and animal facility for excellent support. This work was in part supported by the Lake Road Foundation, the Mr. William H. and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, Stand Up To Cancer/American Association for Cancer Research (a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research), the Lymphoma and Leukemia Society, NYSTEM, NYSCF and the MSK Cancer Center Support Grant/Core Grant (P30 CA008748).
M.S. is a cofounder of and consultant for Juno Therapeutics.
Reviewer Information Nature thanks M. Maus, E. J. Wherry and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Top, TRAC locus with the 5′ end (grey) of the TRAC first exon, the TRAC gRNA (blue) and the corresponding PAM sequence (red). The two blue arrows indicate the predicted Cas9 double strand break. Bottom, CRISPR/Cas9-targeted integration into the TRAC locus. The targeting construct (AAV) contains a splice acceptor (SA), followed by a P2A coding sequence, the 1928z CAR gene and a polyA sequence, flanked by sequences homologous to the TRAC locus (LHA and RHA, left and right homology arm). Once integrated, the endogenous TCRα promoter drives CAR expression, while the TRAC locus is disrupted. TRAV, TCRα variable region; TRAJ, TCRα joining region; 2A, the self-cleaving Porcine teschovirus 2A sequence. pA: bovine growth hormone polyA sequence. b, Timeline of the CAR targeting into primary T cells. c, Representative TCR/CAR flow plots 4 days after transfection of T cells with Cas9 mRNA and TRAC gRNA and addition of AAV6 at the indicated multiplicity of infection. d, Percentage of TCR disruption 4 days post transfection of the Cas9 mRNA and the TRAC gRNA measured by FACS analysis of the TCR expression (n = 5). e, Percentage of knock-in depending on the AAV6 multiplicity of infection measured by FACS analysis of the CAR expression (n = 4). f, Percentage of CAR+ cells in the TCR-negative population (n = 4). g, Percentage of TCR-positive and TCR-negative in the CAR+ population analysed by FACS (n = 4).
Extended Data Figure 2 Whole-genome mapping of the AAV6 TRAC-1928z integration using the TLA technology.
a, Schematic representation of the TLA technology17. For this study, two sets of primers targeting the CAR and the left homology arm have been used. b, TCR/CAR FACS plot of the TRAC-1928z CAR T cells used for the TLA analysis. CAR T cells have been processed as in Extended Data Fig. 1b and expanded for 2 weeks. c, TLA sequence coverage across the human genome using 1928z CAR specific primers (CD28-specific forward: 5′-ACAATGAGAAGAGCAATGGA-3′ and scFV-specific reverse: 5′-GAGATTGTCCTGGTTTCTGT-3′). The chromosomes are indicated on the y axis, the chromosomal position on the x axis. TRAC-encoded CAR T cells were produced as in Fig. 1 and expanded for 10 days before processed for analysis. The primer set was used in an individual TLA amplification. PCR products were purified and library prepped using the Illumina NexteraXT protocol and sequenced on an Illumina Miseq sequencer. Reads were mapped using BWA-SW, which is a Smith–Waterman alignment tool. This allows partial mapping, which is optimally suited for identifying break-spanning reads. The human genome version hg19 was used for mapping. d, TLA sequence coverage aligned on the AAV-TRAC-1928z sequence (Targeting sequence flanked by ITRs). The grey vertical bars on top represent the coverage at the shown positions. The coverage showed integration of the AAV ITRs in fraction of reads. The coverage comparison between ITR and CAR integration at the 5′ and 3′ ends of the TRAC homology arms locus allow the measurement of faithful and unfaithful homologous recombination shown in e. e, Final results from the TLA analysis.
Extended Data Figure 3 In vitro cytotoxicity activity and proliferation response of TRAC-CAR T cells.
a, Representative flow cytometry analysis showing CAR and TCR expression. TRAC-1928z CAR T cells were generated as in Fig. 1b; CRISPR/Cas9-generated TCR– T cells were transduced with RV-1928z retroviral vector; TCR+ cells were transduced with either RV-1928z or RV-P28z (PSMA-specific CAR). TCR-negative T-cell purification was performed using magnetic beads on column. b, Cytotoxic activity using an 18 h bioluminescence assay, using firefly luciferase (FFL)-expressing NALM-6 as targets cells (n = 3 independent experiments on 3 healthy donors). c, Representative cumulative cell counts of CAR T cells upon weekly stimulation with CD19+ target cells. Arrows indicate stimulation time points (n = 3 independent experiments on 3 healthy donors).
a, NALM-6-bearing mice were treated with 2 × 105 (left), 1 × 105 (middle) or 5 × 104 (right) CAR T cells. Tumour burden was quantified weekly over a 100-day period using BLI. Quantification is the average photon count of ventral and dorsal acquisitions per animal at all given time points. Each line represents one mouse. Some groups are pooled from two to three independent experiments from different healthy donors, representing n = 6–20 mice per group. Lower, Kaplan–Meier analysis of survival of mice. b–f, NALM-6-bearing mice were treated with 1 × 105 indicated CAR T cells. At 10 and 17 days after CAR T-cell infusion, 7 mice per group were euthanized and bone marrow cells were collected. CAR T cells and NALM-6 cells were analysed and counted with flow cytometry. b, Representative FACS analysis of tumour cells (CD19+GFP+) in the bone marrow at day 17. c, Representative FACS analysis of exhaustion markers PD1 and TIM3 in bone marrow CAR T cells at day 17. d, Representative FACS analysis of exhaustion markers PD1 and LAG3 in bone marrow CAR T cells at day 17. e, CAR MFI of the CAR+ cells in the bone marrow (each dot represents one mouse). f, Coefficient of variation measuring the dispersion in the CAR expression of the CAR+ population (ratio of the standard deviation to the mean; each dot represents one mouse). g, RV-1928z CAR design allows the co-expression of the CAR and LNGFR from the same LTR promoter by using a self-cleaving P2A sequence. LTR, long terminal repeat, SD, splice donor site; SA, splice acceptor site; 2A, Porcine teschovirus self-cleaving 2A sequence. h, Representative flow cytometry plots of RV-1928z transduced T cells cultured in vitro or in vivo (extracted from bone marrow) and labelled to detect CAR and LNGFR expression. i, Comparison between CAR MFI in the RV-1928z T cells and the tumour burden (NALM-6 count) in the bone marrow.
Extended Data Figure 5 TRAC-19BBz CAR T cells outperform conventional 19BBz CAR T cells by preventing exhaustion in vivo.
a, b, These results compiled the average CAR MFI (a) and coefficient of variation (b) of CAR+ T cells obtained from three independent transfections or transductions. The T cells used for these three experiments have been isolated from blood of three different healthy donors. c, Left, activation, memory, and exhaustion markers of CAR T cells analysed by flow cytometry 5 days after gene transfer. Left, plots indicate the phenotypes of the CAR+ T cells measured by flow cytometry analysis of CD62L and CD45RA expression 5 days after CAR vectorization; colours as in e. d, Relative CAR MFI (1 = MFI at 0 h) after CAR T cells being activated 1, 2 or 4 times on CD19+ target cells over a 48 h periods (n = 3 independent experiments, arrows indicate stimulation time points). e, CAR T cells stimulated on CD19+ target cells either 1, 2 or 4 times in 48 h period were analysed by flow cytometry. Plots indicate the phenotypes of the CAR+ T cells measured by flow cytometry analysis of CD62L and CD45RA expression (average proportion from 3 independent experiments). f, FFL-NALM-6-bearing mice were treated with 1 × 105 CAR T cells. Tumour burden shown as bioluminescent signal quantified per animal every week over a 21-day period. n = 6 mice per group. g–j, NALM-6-bearing mice were treated with 1 × 105 CAR T cells. At 10 and 17 days after CAR T-cell infusion, 7 mice per group were euthanized and bone marrow cells were collected. CAR T cells and NALM-6 cells were analysed and counted with flow cytometry. Each dot represents one mouse. g, CAR T cells count in marrow (n = 7). h, Tumour (CD19+GFP+ NALM-6) cells count in bone marrow (n = 7). i, Effector/tumour ratio in the bone marrow (n = 7). j, Exhaustion marker analysis from bone marrow T cells collected at day 17 and analysed by flow cytometry. Represented as the average percentage of cells expressing the indicated markers (n = 7). *P < 0.05, **P < 0.01, ***P < 0.001 (Mann–Whitney test (a, b) ANOVA F-test (d); see Supplementary information)
Extended Data Figure 6 TRAC-CAR T cells show reduced tonic signalling and antigen-induced differentiation in vitro.
a, Representative FACS analysis of T cells differentiation markers 5 days after the CAR gene transfer. b, Representative FACS analysis of the CAR T cell differentiation markers after 1, 2 or 4 stimulations on CD19+ target cells. c, CAR T cells expansion when stimulated 1, 2 or 4 times on CD19+ target cells over a 48 h period. No noticeable difference in the proliferation was found between the three 1928z CAR T cells conditions. d, Percentage of CAR T cells with positive expression of IFNγ, TNFα or IL-2 after intracellular staining at the end of the protocol in Fig. 2d (n = 2 independent experiments on 2 donors).
Extended Data Figure 7 TRAC-CAR T cells show delayed in vitro antigen-induced differentiantion compared to lowly or highly transduced RV-CAR T cells.
a, Representative histogram of the CAR expression 5 days after transduction of different volume of retroviral supernatant in μl (representative of 3 independent experiments; total transduction volume 2 ml). b, Percentage of CAR+ T cells in function of the volume of retroviral supernatant analysed by FACS 5 days after transduction. (n = 3 donors). c. CAR mean fluorescence intensity (MFI) of T cells as a function of the volume of retroviral supernatant analysed by FACS 5 days after transduction (n = 3 donors). d, CAR coefficient of variation as a function of the volume of retroviral supernatant analysed by FACS 5 days after transduction (n = 3 donors). e, Average CAR MFI of CAR T cells 5 days after transduction (n = 3 donors). High = 1,000 μl, and low = 30 μl. f, CAR T cells stimulated on CD19+ target cells either 1, 2 or 4 times in 48 h period were analysed by flow cytometry. Plots indicate the phenotypes of the CAR-positive T cells measured by flow cytometry analysis of CD62L and CD45RA expression (average proportion from of 3 independent experiments).
Extended Data Figure 8 CAR gene expression using different promoters at distinct loci influences tonic signalling levels in vitro.
a, CRISPR/Cas9-targeted integration into the TRAC locus. The targeting construct (AAV) contains a splice acceptor (SA), followed by a P2A coding sequence, the 1928z CAR gene and a polyA sequence, flanked by sequences homologous to the TRAC locus (LHA and RHA, left and right homology arm). Once integrated, the endogenous TCRα promoter drives CAR expression, while the TRAC locus is disrupted. TRAV, TCRα variable region; TRAJ, TCRα joining region; 2A, the self-cleaving Porcine teschovirus 2A sequence. b, CRISPR/Cas9-targeted promoter integration into the TRAC locus. The targeting construct (AAV) contains the 1928z CAR coding sequence in the reverse orientation, under the control of an exogenous promoter, the long version of human EF1α, the enhancer sequence from the gamma retrovirus used in Figs 1, 2 (Mo-MLV LTR here called LTR) or the phosphoglycerate kinase (PGK) promoter and a polyA sequence, flanked by sequences homologous to the TRAC locus (LHA and RHA, left and right homology arm). c, Schematic of tailored CRISPR/Cas9-induced targeted integration into the B2M locus. The targeting construct (AAV) contains the CAR gene flanked by homology sequences (LHA and RHA). Once integrated, the endogenous B2M promoter drives CAR expression. d, CRISPR/Cas9-targeted promoter integration into the B2M locus. The targeting construct (AAV) contains the 1928z CAR gene in the reverse orientation, under the control of an exogenous promoter, human EF1α, the PGK promoter or a truncated version of the PGK (PGK100) and a polyA sequence, flanked by sequences homologous to the B2M locus (LHA and RHA, left and right homology arm). e, Average CAR mean fluorescence intensity (MFI) analysed by FACS 4 days after transduction (n = 3 to 7 independent experiments and 4 different donors). pA: bovine growth hormone polyA sequence for all targeting constructs. f, Left, representative histogram of the CAR expression 5 days after its vectorization into T cells. Middle, activation, memory, and exhaustion markers of CAR T cells analysed by flow cytometry 5 days after the vectorization of the CAR. Right, plots indicate the phenotypes of the CAR+ T cells measured by flow cytometry analysis of CD62L and CD45RA expression 5 days after CAR gene transfer.
Extended Data Figure 9 CAR gene expression using different promoters at distinct loci influences antigen-induced differentiation and exhaustion in vivo.
a, Representative FACS analysis of the CAR T-cell differentiation markers after 1, 2 or 4 stimulations on CD19+ target cells. b, CAR T-cell expansion when stimulated 1, 2 or 4 times on CD19+ target cells over a 48 h period. No apparent difference in the proliferation was found between the four 1928z CAR T cells conditions. c–e, NALM-6-bearing mice were treated with 1 × 105 CAR T cells. At 10 and 17 days after CAR T cell infusion, 7 mice per group were euthanized and bone marrow cells were collected. CAR T cells and NALM-6 cells were analysed and counted with flow cytometry. Each dot represents one mouse. f, Percentage of effector memory (‘Eff mem’, CD62L−CD45RA−) and effector (‘Eff’, CD62L−CD45RA+) in the bone marrow CAR T cells at day 17 (n = 7 mice). g, Exhaustion marker analysis from bone marrow T cells collected at day 17 and analysed by flow cytometry. Represented as the average percentage of cells expressing the indicated markers (n = 7 mice).
Extended Data Figure 10 Locus-promoter configuration controls CAR protein expression and transcriptional response upon CAR T cell activation.
a, Left, representative histogram of the CAR expression 5 days after its vectorization into T cells. Right, relative CAR MFI (1 = MFI at 0 h) after CAR T cells being activated 1, 2 or 4 times on CD19+ target cells over a 48 h period. b, Comparison between CAR MFI and CAR RNA relative level before stimulation (n = 3 independent experiment on 3 donors).
About this article
Cite this article
Eyquem, J., Mansilla-Soto, J., Giavridis, T. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017). https://doi.org/10.1038/nature21405
Molecular Cancer (2022)
Biomarker Research (2022)
Stem Cell Research & Therapy (2022)
Journal of Hematology & Oncology (2022)
Experimental Hematology & Oncology (2022)