Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 editing of immune checkpoint genes could improve the efficacy of T cell therapy, but the first necessary undertaking is to understand the safety and feasibility. Here, we report results from a first-in-human phase I clinical trial of CRISPR–Cas9 PD-1-edited T cells in patients with advanced non-small-cell lung cancer (ClinicalTrials.gov NCT02793856). Primary endpoints were safety and feasibility, and the secondary endpoint was efficacy. The exploratory objectives included tracking of edited T cells. All prespecified endpoints were met. PD-1-edited T cells were manufactured ex vivo by cotransfection using electroporation of Cas9 and single guide RNA plasmids. A total of 22 patients were enrolled; 17 had sufficient edited T cells for infusion, and 12 were able to receive treatment. All treatment-related adverse events were grade 1/2. Edited T cells were detectable in peripheral blood after infusion. The median progression-free survival was 7.7 weeks (95% confidence interval, 6.9 to 8.5 weeks) and median overall survival was 42.6 weeks (95% confidence interval, 10.3–74.9 weeks). The median mutation frequency of off-target events was 0.05% (range, 0–0.25%) at 18 candidate sites by next generation sequencing. We conclude that clinical application of CRISPR–Cas9 gene-edited T cells is generally safe and feasible. Future trials should use superior gene editing approaches to improve therapeutic efficacy.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All requests for raw and analyzed data and materials are promptly reviewed by the West China Hospital to verify whether the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical trials and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. All other data that support the findings of this study will be provided by the corresponding author upon reasonable request when possible. Raw data for Figs. 2–4 and Extended Data Figs. 1–3, 6 and 8–10 are in the Source Data. The raw sequencing data reported in the study have been deposited in the Genome Sequence Archive for Human (http://bigd.big.ac.cn/gsa-human/) at the BIG Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number PRJCA002488.
Change history
18 June 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41591-020-0973-6
References
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).
Xu, L. et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N. Engl. J. Med. 381, 1240–1247 (2019).
Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).
Mok, T. S. K. et al. Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial. Lancet 393, 1819–1830 (2019).
Reck, M. et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N. Engl. J. Med. 375, 1823–1833 (2016).
Garon, E. B. et al. Five-year overall survival for patients with advanced nonsmall-cell lung cancer treated with pembrolizumab: results from the phase I KEYNOTE-001 Study. J. Clin. Oncol. 37, 2518–2527 (2019).
Su, S. et al. CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer. Oncoimmunology 6, e1249558 (2016).
Beane, J. D. et al. Clinical scale zinc finger nuclease-mediated gene editing of PD-1 in tumor infiltrating lymphocytes for the treatment of metastatic melanoma. Mol. Ther. 23, 1380–1390 (2015).
Chen, W. et al. Cancer statistics in China, 2015. CA Cancer J. Clin. 66, 115–132 (2016).
Bray, F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, 394–424 (2018).
Su, S. et al. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci. Rep. 6, 20070 (2016).
Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 10437–10442 (2015).
Xie, S., Shen, B., Zhang, C., Huang, X. & Zhang, Y. sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PLoS ONE 9, e100448 (2014).
Lv, W. et al. Noninvasive prenatal testing for Wilson disease by use of circulating single-molecule amplification and resequencing technology (cSMART). Clin. Chem. 61, 172–181 (2015).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Manghwar, H. et al. CRISPR/Cas systems in genome editing: methodologies and tools for sgRNA design, off-target evaluation, and strategies to mitigate off-target effects. Adv. Sci. 7, 1902312 (2020).
Robert, L. et al. CTLA4 blockade broadens the peripheral T-cell receptor repertoire. Clin. Cancer Res. 20, 2424–2432 (2014).
Johnson, D. B. et al. Fulminant myocarditis with combination immune checkpoint blockade. N. Engl. J. Med. 375, 1749–1755 (2016).
Sui, T. et al. CRISPR-induced exon skipping is dependent on premature termination codon mutations. Genome Biol. 19, 164 (2018).
Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).
Kalos, M. & June, C. H. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39, 49–60 (2013).
Rapoport, A. P. et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat. Med. 21, 914–921 (2015).
Pasetto, A. et al. Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor. Cancer Imunol. Res. 4, 734–743 (2016).
Zarour, H. M. Reversing T-cell dysfunction and exhaustion in cancer. Clin. Cancer Res. 22, 1856–1864 (2016).
Dijkstra, K. K. et al. Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell 174, 1586–1598.e1512 (2018).
Mandal, P. K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).
Ren, J. et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin. Cancer Res. 23, 2255–2266 (2017).
Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).
Curti, B. D. et al. Phase I trial of anti-CD3-stimulated CD4+ T cells, infusional interleukin-2, and cyclophosphamide in patients with advanced cancer. J. Clin. Oncol. 16, 2752–2760 (1998).
Edge, S. B. et al. AJCC Cancer Staging Manual 7th edn (Springer, 2010).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Tarasov, A., Vilella, A. J., Cuppen, E., Nijman, I. J. & Prins, P. Sambamba: fast processing of NGS alignment formats. Bioinformatics 31, 2032–2034 (2015).
Kim, S. et al. Strelka2: fast and accurate calling of germline and somatic variants. Nat. Methods 15, 591–594 (2018).
Zhou, H. et al. Whole genome analysis of CRISPR Cas9 sgRNA off-target homologies via an efficient computational algorithm. BMC Genomics 18, 826 (2017).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
Wang, Z. et al. Application of single-molecule amplification and resequencing technology for broad surveillance of plasma mutations in patients with advanced lung adenocarcinoma. J. Mol. Diagn. 19, 169–181 (2017).
Shen, B. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11, 399–402 (2014).
Jia, Q. et al. Local mutational diversity drives intratumoral immune heterogeneity in non-small cell lung cancer. Nat. Commun. 9, 5361 (2018).
Acknowledgements
This clinical trial was supported by the 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (grant no. ZYJC18001); the West China Hospital Foundation of New Technology (grant nos. XJS2016003 and 190160012); the Sichuan Cancer Society Foundation (grant no. SCS-KT001); the National Science and Technology Major Project (grant no. 2017ZX09304023); and the National Natural Science Foundation of China (grant no. 81672982). We thank all of the study participants, H. Wakelee and G. P. Gao for providing insightful advice on this study, J.S. Kim for advice on the off-target effects, J.Y. Li and the nursing team for clinical care, Q. Lu for data collection, Q. Zhang for clinical ECG diagnosis, L. Wang for supporting preclinical study, J. Jiang for data and safety monitoring, M. Zhao for data management and S. Wang for statistical support.
Author information
Authors and Affiliations
Contributions
Y. Lu, J.X., L.D. and T.M. were involved in study design. Y. Lu and T.D. contributed to study concepts. T.D., K.Y. and Y. Zeng were responsible for manufacturing of therapeutic cells. X. Zhou, M.H., R.T., Z.D., Y.G., J.Z., Yongsheng Wang, L.L., Y. Zhang, Y. Liu, B.Z., M.Y., L.Z., Y. Li, Q. Z. and B.Y. were involved in data acquisition. Y. Lu, Yu Wang, H.S. and M.L were involved in quality control of data and algorithms. J.X., X. Zhou, X.Y., J.S., J.L., Yuqi Wang, X.S., W.W., X. Zhang, L.Y., X.X. and C.C. were involved in data analysis and interpretation. Yu Wang and H.S. contributed to statistical analysis. Y. Lu, J.X., R.T. and T.M. wrote the manuscript. Yuquan Wei and W.L. were involved in administrative support and supervision. All authors approved the article for submission and publication.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 T7E1 cleavage assay, TA cloning sequencing of PD-1 disruption in cultured T cells.
a, DNA amplified from edited or unedited T cells were subjected to T7E1 cleavage assay. T cells from a healthy person served as a control. The blue arrow indicates the expected bands for uncut (no mismatch) PD-1; the red arrow, expected bands from the T7E1 assay. Marker, DL2000 Marker (Innova GENE Biosciences, Ontario, Canada). b, The efficiency of PD-1 editing was analyzed by TA cloning on day 21 after electroporation.
Extended Data Fig. 2 Long-term effects of PD-1 disruption in cultured T cells in Patient B-01 and C-02.
Viability of PD-1 disruption in long-term cultured T cells. Compared to the rapid decrease in viability of unedited T cells after day 30, the viability of edited T cells was over 90% and remained high until day 40. Total cell numbers of PD-1 disruption in long-term cultured T cells. The numbers of edited T cells increased slowly until around day 30, reflecting delayed proliferation likely due to the electroporation procedure; after day 30, the numbers of edited T cells increased rapidly. By contrast, the numbers of unedited cells, decreased rapidly after day 30.
Extended Data Fig. 3 Off-target analysis by next generation sequencing (NGS).
Characteristics of on-target and off-target mutation types, frequencies and numbers determined by next-generation sequencing (NGS) for the edited T cells of 7 patients prior to the second cycle of infusion. Bar graph and pie graph above represent the types, the numbers and the composition of off-target mutation, color-coded according to the legend in the top-right corner. Intergenic (44.4%) and intronic (39.1%) mutations composed the majority proportion. Heatmap shows the mutation number of predicted off-target sites (18 off-target sites, OT1-18) and on-target site for individuals. Bar graph on right represents mean mutation frequencies of each site among the 7 patients. The mutation frequencies at these off-target sites and the on-target site were 0.05% (range 0.00–0.22%) and 4.09%, respectively. The modification ratio of on-target/off-target was 105.2. Pie graph on bottom-right shows the composition of on-target mutation. The mutation types of on-target consisted of frameshift or nonframeshift (deletion/insertion mutation), and stopgain, while the vast majority was the deletion mutation (88.5%). Data in bar graph are shown as mean ± s.d.
Extended Data Fig. 4 Electrocardiograph and echocardiography of patient Pre-A-01 during treatment.
a, Electrocardiograph images of patient Pre-A-01 during T-cell therapy. Patient Pre-A-01 had no history of heart disease. The baseline electrocardiograph (before infusion) showed normal results. However, the electrocardiograph on day 1 after the first infusion showed a premature beat; the electrocardiograph on the day 113 showed a premature beat similar to that on day 1. Each image is representative of 3 independent tests. b, Representative images from baseline echocardiography (before infusion, left) and echocardiography conducted on day 26 after the first infusion (right). No cardiac lesion or obvious change in functional parameters was found.
Extended Data Fig. 5
Baseline characteristics of all treated patients.
Extended Data Fig. 6 Duration of treatment-related adverse events.
Different colors are used to represent each patient. Bar length represents duration of the adverse effect. All related AEs were grade 1 or 2. Grade 2 AEs are outlined in black.
Extended Data Fig. 7 Follow-up diagram and data.
a, Follow-up diagram after treatment. b, Summary of treatment-related AEs and severe adverse events (SAEs) during follow-up.
Extended Data Fig. 8 Kaplan-Meier estimates of survival in 12 patients.
a, Overall survival. b, Progression-free survival.
Extended Data Fig. 9 TCR diversity in healthy donors and patients.
Comparison of TCR diversity (Shannon index) in PBMCs of 11 healthy donors (n = 11) and 12 patients with refractory NSCLC (n = 12). Data are shown as median ± 95% confidence interval [CI], each dot represents an individual data. P value was calculated using the two-tailed Wilcoxon rank-sum test. Median of differences was -2.457, 95% CI for difference was −4.096 to −1.067, P = 0.0005.
Extended Data Fig. 10 The immunohistochemistry staining density was semi-quantified by ImageJ software.
The data was compared using one-way Anova with Sidak’s multiple comparisons test (n = 3 per group). Data are shown as mean ± s.d., * P < 0.05, ** P < 0.01, ***P < 0.001 and ****P < 0.0001.
Supplementary information
Supplementary Information
Supplementary Fig. 1, Tables 1–8, and Notes
Source data
Source Data Fig. 2
Statistical Source Data
Source Data Fig. 3
Statistical Source Data
Source Data Fig. 4
Statistical Source Data
Source Data Fig. 5
Statistical Source Data
Source Data Extended Data Fig. 1
Unprocessed gels
Source Data Extended Data Fig. 1
Statistical Source Data
Source Data Extended Data Fig. 2
Statistical Source Data
Source Data Extended Data Fig. 3
Statistical Source Data
Source Data Extended Data Fig. 6
Statistical Source Data
Source Data Extended Data Fig. 8
Statistical Source Data
Source Data Extended Data Fig. 9
Statistical Source Data
Source Data Extended Data Fig. 10
Statistical Source Data
Rights and permissions
About this article
Cite this article
Lu, Y., Xue, J., Deng, T. et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med 26, 732–740 (2020). https://doi.org/10.1038/s41591-020-0840-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41591-020-0840-5
This article is cited by
-
CRISPR/Cas-based CAR-T cells: production and application
Biomarker Research (2024)
-
CircRNA as an Achilles heel of cancer: characterization, biomarker and therapeutic modalities
Journal of Translational Medicine (2024)
-
CRISPR–Cas9 applications in T cells and adoptive T cell therapies
Cellular & Molecular Biology Letters (2024)
-
Correction: Comprehensive review of CRISPR‑based gene editing: mechanisms, challenges, and applications in cancer therapy
Molecular Cancer (2024)
-
Harnessing the evolving CRISPR/Cas9 for precision oncology
Journal of Translational Medicine (2024)
