Mailin Li

Introduction. Chronic Lymphocytic Leukemia (CLL) is a cancer of mature B-cells and is the predominant leukemia affecting Western countries.¹ Chimeric antigen receptor T-cell (CAR-T) therapy is a promising alternative treatment for many B-cell malignancies including CLL, especially for patients refractory or relapsed to standard treatments.¹ In CAR-T therapy, T-cells are isolated from the patient, transduced with a virus overexpressing a recombinant T-cell receptor, and re-infused into the patient to execute a T-cell-mediated killing of tumor cells.²³⁴ Early clinical trials showed CAR-T treatment for CLL resulting in variable patient responsiveness.⁵ A non-homogenous CAR-T population, both as consequence of random viral integration and antigen-dependent changes within the patient post-infusion, are potential mechanisms explaining patient-to-patient inconsistencies. Thus, transgene integration into a specific genomic locus is currently being explored to standardize CAR-T effectiveness and avoid the state of T-cell exhaustion and reduced cell killing often induced by chronic antigen stimulation as in the setting of cancer. Methods. Integration of the CAR-T transgene in the Ten-Eleven-Translocation 2 (TET2) locus achieved durable remission in a 78-year-old man with advanced relapsed/refractory CLL.² Patient peripheral blood was isolated over the course of treatment and sequenced to identify the CAR transgene integration site, monitor tumor burden, and track T-cell clonotype frequencies and proliferation. Because TET2 is an active demethylase regulating T-cell differentiation and effector function genes via DNA demethylation, Assay-for-Transposase-Accessible-Chromatin-using-Sequencing (ATAC-Seq) was used to identify regions of altered chromatin accessibility after TET2 disruption. Finally, flow cytometry was performed to assess changing distributions of T-cell phenotypes and their contribution to the anti-tumor response. Results. Sequencing of the T-cell receptor β chain identified clone TCRVβ5.1 as the dominant CAR-T clone at the height of the anti-tumor response, with CAR integration disrupting one TET2 allele and a hypomorphic mutation on the second allele. As revealed by ATAC-Seq, loss of TET2 activity revealed more closed chromatin regions in genes regulating T-cell effector functions, differentiation, and exhaustion due to prolonged stimulation. Additionally, more open chromatin signatures in cell cycle and T-cell receptor signaling genes were observed with loss of TET2. Flow cytometry showed that TET2 disruption resulted in an enlarged central memory T-cell compartment, which dramatically expanded in vivo to facilitate a sustained anti-tumor response. An in vitro re-stimulation assay evaluating T-cell exhaustion further showed that primary T-cells transduced with TET2 shRNA were able to sustain proliferation when serially stimulated with tumor antigen, compared to control transduced T-cells that became nonresponsive over time.  Conclusions. Overall, disruption of TET2 favored the development of CAR-T cells with a highly proliferative, less differentiated but more exhaustion-resistant phenotype that translated into significant therapeutic effect in treating one and potentially many additional CLL patients.

1. Burger JA. Treatment of Chronic Lymphocytic Leukemia. N Engl J Med. 2020;383(5):460-473. doi:10.1056/NEJMra1908213
2. Fraietta JA, Nobles CL, Sammons MA, et al. Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature. 2018;558(7709):307-312. doi:10.1038/s41586-018-0178-z
3. Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol. 2020;20(11):651-668. doi:10.1038/s41577-020-0306-5
4. June CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med. 2018;379(1):64-73. doi:10.1056/NEJMra1706169
5. Fraietta JA, Lacey SF, Orlando EJ, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018;24(5):563-571. doi:10.1038/s41591-018-0010-1