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Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

ABSTRACT: Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19⁺ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19⁺ tumors in an in vivo mouse model.

SUBMITTER: MacLeod DT

PROVIDER: S-EPMC5383629 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

MacLeod Daniel T DT Antony Jeyaraj J Martin Aaron J AJ Moser Rachel J RJ Hekele Armin A Wetzel Keith J KJ Brown Audrey E AE Triggiano Melissa A MA Hux Jo Ann JA Pham Christina D CD Bartsevich Victor V VV Turner Caitlin A CA Lape Janel J Kirkland Samantha S Beard Clayton W CW Smith Jeff J Hirsch Matthew L ML Nicholson Michael G MG Jantz Derek D McCreedy Bruce B

Molecular therapy : the journal of the American Society of Gene Therapy 20170223 4

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19<sup>+</sup> hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease stat ...[more]

PMID: 28237835

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Project description:BackgroundCD4 T lymphocyte activation requires T cell receptor (TCR) engagement by peptide/MHC (major histocompatibility complex) (pMHC). The TCR complementarity-determining region 3 (CDR3) contains variable ? and ? loops critical for pMHC recognition. During any immune response, tuning of TCR usage through progressive clonal selection occurs. Th1 and Th2 cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. We therefore hypothesized that CDR3?? sequence features may intrinsically influence CD4 phenotype during progression of a response.ResultsWe show that CD4 polarization involves distinct CDR3? usage: Th1 and Th17 cells favored short TCR CDR3? sequences of 12 and 11 amino acids, respectively, while Th2 cells favored elongated CDR3? loops of 14 amino acids, with lower predicted affinity. The dominant Th2- and Th1-derived TCR? sequences with 14 amino acid CDR3 loops and 12 amino acid CDR3 loops, respectively, were expressed in TCR transgenics. The functional impact of these TCR? transgenes was assessed after in vivo priming with a peptide/adjuvant. The short, Th1-derived receptor transgenic T cell lines made IFN?, but not IL-4, 5 or 13, while the elongated, Th2-derived receptor transgenic T cell lines made little or no IFN?, but increased IL-4, 5 and 13 with progressive re-stimulations, mirrored by GATA-3 up-regulation. T cells from primed Th2 TCR? transgenics selected dominant TCR V? expansions, allowing us to generate TCR?? transgenics carrying the favored, Th2-derived receptor heterodimer. Primed T cells from TCR?? transgenics made little or no IL-17 or IFN?, but favored IL-9 after priming with Complete Freund's adjuvant and IL-4, 5, 9, 10 and 13 after priming with incomplete Freund's. In tetramer-binding studies, this transgenic receptor showed low binding avidity for pMHC and polarized T cell lines show TCR avidity for Th17 > Th1 > Th2. While transgenic expression of a Th2-derived, 'elongated' TCR-CDR3? and the TCR?? pair, clearly generated a program shifted away from Th1 immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose.ConclusionWe propose that selection from responding clones with distinctive TCRs on the basis of functional avidity can direct a preference away from Th1 effector responses, favoring Th2 cytokines.

Dataset Information

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

Publications

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

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