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TCRαβ/CD3 disruption enables CD3-specific antileukemic T cell immunotherapy
Jane Rasaiyaah, … , Ulrike Mock, Waseem Qasim
Jane Rasaiyaah, … , Ulrike Mock, Waseem Qasim
Published July 12, 2018
Citation Information: JCI Insight. 2018;3(13):e99442. https://doi.org/10.1172/jci.insight.99442.
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Research Article Cell biology Immunology

TCRαβ/CD3 disruption enables CD3-specific antileukemic T cell immunotherapy

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Abstract

T cells engineered to express chimeric antigen receptors (CARs) against B cell antigens are being investigated as cellular immunotherapies. Similar approaches designed to target T cell malignancies have been hampered by the critical issue of T-on-T cytotoxicity, whereby fratricide or self-destruction of healthy T cells prohibits cell product manufacture. To date, there have been no reports of T cells engineered to target the definitive T cell marker, CD3 (3CAR). Recent improvements in gene editing now provide access to efficient disruption of such molecules on T cells, and this has provided a route to generation of 3CAR, CD3-specific CAR T cells. T cells were transduced with a lentiviral vector incorporating an anti-CD3ε CAR derived from OKT3, either before or after TALEN-mediated disruption of the endogenous TCRαβ/CD3 complex. Only transduction after disrupting assembly of TCRαβ/CD3 yielded viable 3CAR T cells, and these cultures were found to undergo self-enrichment for 3CAR+TCR–CD3– T cells without any further processing. Specific cytotoxicity against CD3ε was demonstrated against primary T cells and against childhood T cell acute lymphoblastic leukemia (T-ALL). 3CAR T cells mediated potent antileukemic effects in a human/murine chimeric model, supporting the application of cellular immunotherapy strategies against T cell malignancies. 3CAR provides a bridging strategy to achieve T cell eradication and leukemic remission ahead of conditioned allogeneic stem cell transplantation.

Authors

Jane Rasaiyaah, Christos Georgiadis, Roland Preece, Ulrike Mock, Waseem Qasim

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Figure 1

Successful generation of 3CAR requires disruption of CD3ε expression prior to lentiviral 3CAR transduction.

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Successful generation of 3CAR requires disruption of CD3ε expression pri...
(A) Vector configuration showing codon optimized single-chain variable fragment (scFv) derived from OKT3 with CD8 stalk and 41BB/CD3ζ activation domains, all under the control of an internal human phosphoglycerate kinase (hPGK) promoter in a third-generation self-inactivating (SIN) lentivirus with HIV-1–derived reverse response element (RRE), central polypurine tract (cPPT), and mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Human cytomegalovirus (CMV); Repeat region (R); Unique 5’ region (U5); Psi (Ψ); and modified unique 3’ region (ΔU3). (B) Percentage of CAR+ cells with and without TRAC-KO prior to lentivirus (LV). Data are presented as mean ± SEM. n = 3 donors. (C) Schema of event precedence required for successful 3CAR T cell production. CD3/CD28 activation with TransAct for 48 hours was followed by electroporation of mRNA encoding TRAC-specific TALENs ahead of LV transduction by 72 hours. (D) Summary of CD3+ cells following TRAC-KO (n = 3 donors), mean ± SEM. ***P < 0.0005, by unpaired, 2-tailed Student’s t test. (E) Representative flow cytometry plots of day 7 cells. In cultures with 3CAR expression, there were no surviving CD3+ cells compared with 20% residual expression in the absence of 3CAR transduction (n = 3 donors). (F) Representative flow cytometry plots showing 3CAR T cells retained CD4 and CD8, but not TCRαβ, expression; n = 3 donors. (G) For comparison, control transductions with LV CAR19 mediated 65% transduction but required further processing by column-mediated TCRαβ depletion to yield TRAC-KO CAR19+ T cells; n = 3 donors. (H) Summary of CAR+ cells following TRAC-KO (n = 3 donors); mean ± SEM. Two-tailed Student’s t test.

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