T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant “off-the-shelf” CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant “off-the-shelf” CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin’s T cell lymphoma without a requirement for autologous T cells.
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Ma H, Abdul-Hay M. T-cell lymphomas, a challenging disease: types, treatments, and future. Int J Clin Oncol. 2017;22:18–51.
Karrman K, Johansson B. Pediatric T-cell acute lymphoblastic leukemia. Genes Chromosomes Cancer. 2017;56:89–116.
Gökbuget N, Arnold R, Böhme A, Fietkau R, Freund M, Ganser A. et al. Treatment of adult ALL according to protocols of the German Multicenter Study Group for adult ALL (GMALL). Editors: Estey EH, Faderl SH, Kantarjian HM. Acute leukemias. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008. p. 167–76.
Marks DI, Paietta EM, Moorman AV, Richards SM, Buck G, DeWald G, et al. T-cell acute lymphoblastic leukemia in adults: clinical features, immunophenotype, cytogenetics, and outcome from the large randomized prospective trial (UKALL XII/ECOG 2993). Blood. 2009;114:5136–45.
Goldberg JM, Silverman LB, Levy DE, Dalton VK, Gelber RD, Lehmann L, et al. Childhood T-cell acute lymphoblastic leukemia: the Dana-Farber Cancer Institute acute lymphoblastic leukemia consortium experience. J Clin Oncol. 2003;21:3616–22.
Litzow MR, Ferrando AA. How I treat T-cell acute lymphoblastic leukemia in adults. Blood. 2015;126:833–41.
Porter DL, Hwang W-T, Frey NV, Lacey SF, Shaw PA, Loren AW, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7:303ra139–303ra139.
Kochenderfer JN, Dudley ME, Kassim SH, Somerville RPT, Carpenter RO, Stetler-Stevenson M, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol. 2015;33:540–9.
Park JH, Geyer MB, Brentjens RJ. CD19-targeted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical outcomes to date. Blood. 2016;127:3312–20.
Campana D, Behm FG. Immunophenotyping of leukemia. J Immunol Methods. 2000;243:59–75.
Khalidi HS, Chang KL, Medeiros LJ, Brynes RK, Slovak ML, Murata-Collins JL, et al. Acute lymphoblastic leukemia. Survey of immunophenotype, French-American-British classification, frequency of myeloid antigen expression, and karyotypic abnormalities in 210 pediatric and adult cases. Am J Clin Pathol. 1999;111:467–76.
Patel JL, Smith LM, Anderson J, Abromowitch M, Campana D, Jacobsen J, et al. The immunophenotype of T-lymphoblastic lymphoma in children and adolescents: a Children’s Oncology Group report. Br J Haematol. 2012;159:454–61.
Milush JM, Long BR, Snyder-Cappione JE, Cappione AJ 3rd, York VA, Ndhlovu LC, et al. Functionally distinct subsets of human NK cells and monocyte/DC-like cells identified by coexpression of CD56, CD7, and CD4. Blood. 2009;114:4823–31.
Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, Suhoski MM, et al. Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA. 2009;106:3360–5.
Roguska MA, Pedersen JT, Keddy CA, Henry AH, Searle SJ, Lambert JM, et al. Humanization of murine monoclonal antibodies through variable domain resurfacing. Proc Natl Acad Sci USA. 1994;91:969–73.
Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998;72:8463–71.
Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol. 1998;72:9873–80.
Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015;33:985–9.
Jedema I, Barge RM, Willemze R, Falkenburg JH. High susceptibility of human leukemic cells to Fas-induced apoptosis is restricted to G1 phase of the cell cycle and can be increased by interferon treatment. Leukemia. 2003;17:576–84.
Uy GL, Rettig MP, Motabi IH, McFarland K, Trinkaus KM, Hladnik LM, et al. A phase 1/2 study of chemosensitization with the CXCR4 antagonist plerixafor in relapsed or refractory acute myeloid leukemia. Blood. 2012;119:3917–24.
Rettig MP, Ritchey JK, Prior JL, Haug JS, Piwnica-Worms D, DiPersio JF. Kinetics of in vivo elimination of suicide gene-expressing T cells affects engraftment, graft-versus-host disease, and graft-versus-leukemia after allogeneic bone marrow transplantation. J Immunol. 2004;173:3620–30.
Gross S, Piwnica-Worms D. Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat Methods. 2005;2:607–14.
Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187–97.
Festing MF, Altman DG. Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR J. 2002;43:244–58.
Mead R. The Design of Experiments: Statistical Principles for Practical Applications. Cambridge: Cambridge University Press. 1988.
Eissenberg LG, Rettig MP, Ritchey JK, Prior JL, Schwarz SW, Frye J, et al. [18F]FHBG PET/CT imaging of CD34-TK75 transduced donor T cells in relapsed allogeneic stem cell transplant patients: safety and feasibility. Mol Ther. 2015;23:1110–22.
Png YT, Vinanica N, Kamiya T, Shimasaki N, Coustan-Smith E, Campana D. Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies. Blood Adv. 2017;1:2348–60.
Gomes-Silva D, Srinivasan M, Sharma S, Lee CM, Wagner DL, Davis TH, et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood. 2017;130:285–96.
Mamonkin M, Rouce RH, Tashiro H, Brenner MK. A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood. 2015;126:983–92.
Osborn MJ, Webber BR, Knipping F, Lonetree CL, Tennis N, DeFeo AP, et al. Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases. Mol Ther. 2016;24:570–81.
Qasim W, Zhan H, Samarasinghe S, Adams S, Amrolia P, Stafford S, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med. 2017;9:eaaj2013.
Pinz K, Liu H, Golightly M, Jares A, Lan F, Zieve GW, et al. Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells. Leukemia. 2016;30:701–7.
Tiftik N, Bolaman Z, Batun S, Ayyildiz O, Isikdogan A, Kadikoylu G, et al. The importance of CD7 and CD56 antigens in acute leukaemias. Int J Clin Pract. 2004;58:149–52.
Miwa H, Nakase K, Kita K. Biological characteristics of CD7(+) acute leukemia. Leuk Lymphoma. 1996;21:239–44.
Bonilla FA, Kokron CM, Swinton P, Geha RS. Targeted gene disruption of murine CD7. Int Immunol. 1997;9:1875–83.
Reinhold U, Liu L, Sesterhenn J, Abken H. CD7-negative T cells represent a separate differentiation pathway in a subset of post-thymic helper T cells. Immunology. 1996;89:391–6.
Stock W, Sanford B, Lozanski G, Vij R, Byrd JC, Powell BL, et al. Alemtuzumab can be incorporated into front-line therapy of adult acute lymphoblastic leukemia (ALL): final phase I results of a Cancer and Leukemia Group B Study (CALGB 10102). Blood. 2009;114:345–345.
Gökbuget N, Basara N, Baurmann H, Beck J, Brüggemann M, Diedrich H, et al. High single-drug activity of nelarabine in relapsed T-lymphoblastic leukemia/lymphoma offers curative option with subsequent stem cell transplantation. Blood. 2011;118:3504–11.
Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529:490–5.
Eyquem J, Mansilla-Soto J, Giavridis T, van der Stegen SJC, Hamieh M, Cunanan KM, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543:113–7.
MacLeod DT, Antony J, Martin AJ, Moser RJ, Hekele A, Wetzel KJ, et al. Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR T cells. Mol Ther. 2017;25:949–61.
Cooke KR, Kobzik L, Martin TR, Brewer J, Delmonte J Jr, Crawford JM, et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood. 1996;8:3230–9.
This work is dedicated in memory of Gordon S. Cooper. 1946–2017. We thank Dr. Carl June (University of Pennsylvania) for providing the backbone of a third-generation CAR and the pELNS-Ef1α lentiviral vector.
Specialized Program of Research Excellence (SPORE) in Leukemia NIH: 1P50CA171063-01A1, R35 CA210084-01A, the Gabrielle’s Angels Foundation, the Children's Discovery Institute of Washington University and St. Louis Children's Hospital, the Alvin J. Siteman Cancer Research Fund at Washington University in St. Louis, MO.
MLC and JFD conceived project. MLC, JFD, JC, and MR designed the experiments. MLC, JKR, JMN, and KE cloned the CAR constructs and generated virus. MLC performed gene editing and generated CAR-T. MLC, JKR, and JO preformed and analyzed in vitro assays. MLC, JKR, JMN, BW, and LNG performed in vivo experiments. JLP and SA performed BLI imaging. KS and MLC performed FACS analysis. DMW and AG developed PDX models. MLC, CAM, CCF, and RSF completed and analyzed off-target nuclease activity analysis. FG performed all statistical analyses. All authors were involved in the interpretation of data and preparation of this manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
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Cooper, M.L., Choi, J., Staser, K. et al. An “off-the-shelf” fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia 32, 1970–1983 (2018). https://doi.org/10.1038/s41375-018-0065-5
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