Long Terminal Repeat CRISPR-CAR-Coupled “Universal” T Cells Mediate Potent Anti-leukemic Effects

Abstract

Gene editing can be used to overcome allo-recognition, which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such "universal" T cells expressing chimeric antigen receptors (CARs) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). Although relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. We describe a self-inactivating lentiviral "terminal" vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the ΔU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR (>99%) TCR populations by automated magnetic separation. Molecular analyses, including NGS, WGS, and Digenome-seq, verified on-target specificity with no evidence of predicted off-target events. Robust anti-leukemic effects were demonstrated in humanized immunodeficient mice and were sustained longer than by conventional CARTCR T cells. Terminal-TRAC (TT) CAR T cells offer the possibility of a pre-manufactured, non-HLA-matched CAR cell therapy and will be evaluated in phase 1 trials against B cell malignancies shortly.

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

“…The Jurkat T cell targets had been stably transduced to express both EGFP and luciferase and, following TALEN-mediated TCRαβ disruption, had been sorted for TCR + and TCRexpressers. Tumor engraftment was confirmed by in vivo imaging of bioluminescence using an IVIS Lumina III In Vivo Imaging System (PerkinElmer, live image version 4.5.18147) on day 3 (19). TCR + -or TCR --injected mice were further injected on day 4 with either PBS (n = 2), 10 × 10 6 untransduced T cells (n = 4), or 10 × 10 6 3CAR T cells (n = 5) per tumor group.…”

Mechanisms of resistance to CAR T cell therapy

“…The results of these early studies demonstrate the feasibility of this approach, with only a low frequency of high-grade graft-versus-host disease (GVHD) [59][60][61] . Manufacturing CAR T cells from third-party donors might enable the development of universal, off-theshelf products and is another method to overcome the problem of quantitatively insufficient or poor-quality CAR T cell products 62,63 . Generating such products will probably require additional genetic modification to limit CAR T cell rejection and/or GVHD but also offers opportunities to overcome resistance mechanisms, such as 'fratricide' reported with T cell antigen-targeted CAR T cell products 64 .…”

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

“…To minimize GVHD and TCR mispairing, TCR components can also be targeted [58,279,286,287]. For example, Eyquem et al directed the insertion of a CAR in the TCRα locus, knocking down the endogenous TCR expression [286].…”