Improved CAR T‑cell Therapies by Targeting the Epigenome

A rapidly progressing immunotherapy approach is called adoptive cell transfer (ACT), where the patient's own immune cells are collected and used to treat their cancer. In CAR T‑cell therapy, the patient's T cells are harvested and engineered to produce chimeric antigen receptors, or CARs, on their surface enabling them to bind to specific tumour antigens. CAR T‑cells are then expanded and infused back into the patient where they detect and kill cancer cells expressing the target antigen.

However, the effectiveness of treatments involving CAR T‑cells has been linked to the persistence and proliferation of the CAR T‑cells in the patient's body and this can be affected by factors including cancer sub-type, molecular design of the CAR used and even the cell's manufacturing process.

Fraietta et al., reported an unusually delayed response from one patient during a clinical trial in chronic lymphocytic leukemia (CLL) using CAR T‑cells targeted to CD‑19 that may shed light on some of the mechanisms that determine the persistence of CAR T‑cells and their clinical outcome.

The patient, 78 year old male with relapsed/refractory CLL, underwent a first round of CAR T‑cell therapy but was diagnosed with cytokine release syndrome (CRS) that required treatment with IL‑6 receptor blocking therapy. Six weeks post CAR‑T infusion the patient still had progressive leukaemia and, amid concerns that IL‑6 blockade attenuated the CAR‑T therapy, the patient received their remaining CAR T‑cells 70 days post first dose. Again CRS was an issue but no IL‑6 intervention was required. The patient's bone marrow still had extensive infiltration of CLL one month after the second infusion and CT scans demonstrated minimal effects on extensive adenopathy, however, two months after the second infusion, the patient had unexpectedly responded to treatment.

Expanded CAR T‑cells were observed to be at peak level in the peripheral blood of the patient 2 months after the second infusion. Following the CAR‑T expansion the patient experienced high-grade CRS with a high fever followed by a rapid clearance of CLL.

A log fold reduction in tumour burden was detected 51 days post second infusion with no detection of CLL in the patient's blood one month later. The patient showed complete remission with no signs of CLL in his bone marrow and no abnormal adenopathy after six months. The patient remains well in complete remission that has been sustained for more than 5 years so far.

Analysis of the T‑Cell beta receptors of the expanded CAR T‑cells showed that the cells almost exclusively belonged to a clonal population descended from a single CAR T‑cell. This single cell's progeny divided (approximately 29 population doublings) over time until they reached the tipping point that eliminated the entire tumour.

To establish why the specific CAR T‑cell worked in such remarkably effective minimal dose, the authors analysed the integration site of the CAR vector. In the clonal population of T‑cells, the CAR sequence has inserted into a copy of the TET2 gene (on intron 9), preventing expression of a functional protein.

The TET2 gene from this patient contained a missense (E1879Q) mutation such that the patient's T‑cells had one allele interrupted by the lentiviral insertion and the other copy of TET2 with a missense mutation. TET2 encodes methylcytosine dioxygenase, an enzyme that mediates demethylation of DNA and is associated with epigenetic modifications that can effect gene expression. Investigation of the mutant when expressed in HEK293 cells revealed attenuated levels of 5‑mC oxidation compared to the WT.

The effect of mutant TET2 on T‑cell function was assessed by checking regions of accessible chromatin using ATAC‑Seq. Although comparison of the overall epigenetic profile of patient's CAR- and CAR+T cell, showed similar epigenetic profile, differences between samples were observed in the structure of chromatin in regions involved in cell cycle and T‑cells receptor signaling.

Loss of TET2 on both alleles caused reduction of peak-binding at sites involved in T‑cell differentiation such as NOTCH2, PRDM1, CD28, ICOS and Interferon‑γ. Cultured T‑cells from the patient expressed less Interferon‑γ and CD107a when activated suggesting impaired differentiation.

Ex vivo CTL019 cells from this patient were analysed for differentiation status compared to six patients who also responded to CAR T‑cell therapy but did not have TET2 disruption. 65% of cells in the TET2 disrupted CAR T‑cells had a central memory type T‑cell phenotype, whereas the other responders had a CD8+ and CTL019 effector memory phenotype.

TET2 inhibition was analysed further by repeated stimulation of CAR T‑cells with CD19 expressing tumour cells. T‑cells lacking TET2 expressed cytokines such as IFNγ and TNFα at lower levels, following antigen stimulation. They also had increased expression of perforin and granzyme B, which are components of the tumour-killing machinery of T‑cells. The clinically active CAR T‑cells were also expressing CD27, a co-stimulatory receptor involved in generation of T‑cell memory. A high frequency of Ki‑67 positive CAR T‑cells were also observed at the peak of in vivo expansion in the patient suggesting that TET2 is required for CAR T‑cell proliferation.

These remarkable findings might suggest that TET2 dysfunction may produce potent CAR T‑cells that can proliferate and persist to eliminate the target tumors. Targeting TET2 in human CAR T‑cells through drug mediated inhibition or gene–editing techniques could increase the effectiveness of CAR T‑cell treatment for other patients. If only small numbers of modified CAR T‑cells are required for effective treatment this would also substantially speed up the waiting times and lower the manufacturing costs of the treatment leading to overall improvement of immunotherapies.

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Epistem's CAR T‑cell Capabilities

Epistem has extensive experience handling CAR T‑cells and offers several pre-clinical human cancer models to assess the efficacy of CAR T‑cell therapies. Epistem has in vivo optical imaging capabilities and can offer orthotopic imaging models of cancer to study the efficacy of CAR T‑cell therapy in the same animal over time. We also have an in-house eight colour MACS quant to evaluate treatment effects and T‑cell function ex vivo.

All our models are supported by GCLP‑compliant histology/IHC and genomic analytical services. We have cell sorting abilities and are able to perform genetic analysis from single cell. We have in-house bioinformatics support for all of our gene expression studies and have extensive experience identifying biomarkers or biomarker signatures in a variety of tissues including skin.

“Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells” Fraietta et al., Nature; 14 June 2018, 558 (7709): 307-312.doi:10.1038/s41586-018-0178-z

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