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

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.

T-cells “à la CAR-T(e)” – Genetically engineering T-cell response against cancer

“…It is also possible to use genome editing to abrogate the expression of the targeted antigen from the (targeting) T cells themselves. Rasaiyaah et al used TALEN to eliminate the endogenous TCR complex while expressing simultaneously an anti-CD3 CAR to target leukemic T cells with minimal off-target effects [290]. Similarly, CD33 expression in progenitor cells was successfully abrogated using CRISPR/Cas9 gene editing [291].…”

Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions

“…Allogeneic CAR T cells with TRAC locus editing [46,61] Using NK cells or NK-92 cells [47,[51][52][53][54]60] Using γδ T cells in vitro cytotoxicity against two T-ALL cell lines and primary T-ALL cells and delayed leukemia progression in two different CD5-positive T-ALL models [59]. Based on these results, CD5-CAR T cells with a CD28 costimulatory domain are being tested in patients with relapsed or refractory T cell disease (MAGENTA trial, NCT03081910).…”

“…As CD3 is exclusively expressed on T cells, it has been a popular target in preclinical CAR T cell therapies for the treatment of T cell malignancies. As expected, due to fratricidal issues, manufacturing of anti-CD3 CAR T cells does not yield a viable cellular product [61]. Various approaches using an anti-CD3 CAR have been investigated including the use of transcription activator-like effector nuclease (TALEN) mRNA to disrupt the TRAC locus and using NK-92 cells in place of T cells as the effector cell type.…”

“…In patient T-ALL samples, anti-CD3 CAR T cells demonstrated specific cytotoxicity against CD3positive cells. In a T-ALL NSG model, anti-CD3 CAR T cells were shown to clear luciferase-expressing CD3positive Jurkat cells, but showed no effect in NSG mice engrafted with CD3-negative Jurkat cells [61]. To circumvent the need for additional modifications, NK-92 cells can also be used to express the anti-CD3-CAR, since they are CD3-negative cells.…”

“…This strategy may be useful to reduce allo-recognition in patients receiving CAR T cell therapy. Other groups have exploited similar approaches in preclinical CAR T cell investigations targeting CD7 and CD3, as previously described [46,61].…”